US20130253048A1 - Pharmaceutical Composition for Promoting Osteogenesis Containing Axial-equatorial Aryl-oriented Furofuran-type Lignan, and Pharmaceutical Preparation, Functional Food Product, and Health Food Product Comprising Composition - Google Patents
Pharmaceutical Composition for Promoting Osteogenesis Containing Axial-equatorial Aryl-oriented Furofuran-type Lignan, and Pharmaceutical Preparation, Functional Food Product, and Health Food Product Comprising Composition Download PDFInfo
- Publication number
- US20130253048A1 US20130253048A1 US13/739,340 US201313739340A US2013253048A1 US 20130253048 A1 US20130253048 A1 US 20130253048A1 US 201313739340 A US201313739340 A US 201313739340A US 2013253048 A1 US2013253048 A1 US 2013253048A1
- Authority
- US
- United States
- Prior art keywords
- group
- bone
- composition
- fargesin
- food product
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Abandoned
Links
- 239000000203 mixture Substances 0.000 title claims abstract description 81
- 239000000825 pharmaceutical preparation Substances 0.000 title claims abstract description 31
- 235000013376 functional food Nutrition 0.000 title claims abstract description 22
- 230000001737 promoting effect Effects 0.000 title claims abstract description 18
- 239000008194 pharmaceutical composition Substances 0.000 title claims description 14
- 235000013402 health food Nutrition 0.000 title abstract description 13
- 230000011164 ossification Effects 0.000 title description 30
- 229930013686 lignan Natural products 0.000 title description 3
- 150000005692 lignans Chemical class 0.000 title description 3
- 235000009408 lignans Nutrition 0.000 title description 3
- 150000003839 salts Chemical class 0.000 claims abstract description 9
- 241000218377 Magnoliaceae Species 0.000 claims abstract description 8
- 239000004480 active ingredient Substances 0.000 claims abstract description 7
- 229930182470 glycoside Natural products 0.000 claims abstract description 7
- 150000002338 glycosides Chemical class 0.000 claims abstract description 7
- 241000196324 Embryophyta Species 0.000 claims abstract description 5
- 239000000126 substance Substances 0.000 claims description 41
- 150000001875 compounds Chemical class 0.000 claims description 32
- 210000000056 organ Anatomy 0.000 claims description 12
- 235000010469 Glycine max Nutrition 0.000 claims description 11
- 235000012149 noodles Nutrition 0.000 claims description 10
- 230000004072 osteoblast differentiation Effects 0.000 claims description 10
- OKTJSMMVPCPJKN-UHFFFAOYSA-N Carbon Chemical compound [C] OKTJSMMVPCPJKN-UHFFFAOYSA-N 0.000 claims description 9
- 240000005378 Magnolia kobus Species 0.000 claims description 9
- 235000014196 Magnolia kobus Nutrition 0.000 claims description 9
- 229910052799 carbon Inorganic materials 0.000 claims description 9
- 239000000284 extract Substances 0.000 claims description 9
- 244000299461 Theobroma cacao Species 0.000 claims description 8
- 235000009508 confectionery Nutrition 0.000 claims description 8
- 240000005819 Magnolia denudata Species 0.000 claims description 6
- 235000016094 Magnolia denudata Nutrition 0.000 claims description 6
- 241000209140 Triticum Species 0.000 claims description 6
- 235000021307 Triticum Nutrition 0.000 claims description 6
- 125000000217 alkyl group Chemical group 0.000 claims description 6
- 235000013353 coffee beverage Nutrition 0.000 claims description 6
- 125000000524 functional group Chemical group 0.000 claims description 6
- 235000013618 yogurt Nutrition 0.000 claims description 6
- 235000001497 healthy food Nutrition 0.000 claims description 5
- 240000007154 Coffea arabica Species 0.000 claims description 4
- 240000008620 Fagopyrum esculentum Species 0.000 claims description 4
- 235000009419 Fagopyrum esculentum Nutrition 0.000 claims description 4
- 241000007358 Magnolia biondii Species 0.000 claims description 4
- 240000007594 Oryza sativa Species 0.000 claims description 4
- 235000007164 Oryza sativa Nutrition 0.000 claims description 4
- 235000009470 Theobroma cacao Nutrition 0.000 claims description 4
- 235000013334 alcoholic beverage Nutrition 0.000 claims description 4
- 235000015895 biscuits Nutrition 0.000 claims description 4
- 235000013339 cereals Nutrition 0.000 claims description 4
- 235000013351 cheese Nutrition 0.000 claims description 4
- 235000019219 chocolate Nutrition 0.000 claims description 4
- 235000016213 coffee Nutrition 0.000 claims description 4
- 235000013365 dairy product Nutrition 0.000 claims description 4
- 235000015094 jam Nutrition 0.000 claims description 4
- 235000010746 mayonnaise Nutrition 0.000 claims description 4
- 239000008268 mayonnaise Substances 0.000 claims description 4
- 235000019520 non-alcoholic beverage Nutrition 0.000 claims description 4
- 235000015927 pasta Nutrition 0.000 claims description 4
- 235000009566 rice Nutrition 0.000 claims description 4
- 235000011888 snacks Nutrition 0.000 claims description 4
- 241001609968 Magnolia praecocissima Species 0.000 claims description 3
- 125000003545 alkoxy group Chemical group 0.000 claims description 3
- 125000004435 hydrogen atom Chemical group [H]* 0.000 claims description 3
- 125000002887 hydroxy group Chemical group [H]O* 0.000 claims description 3
- 241000218296 Magnolia salicifolia Species 0.000 claims description 2
- 241000007370 Magnolia sprengeri Species 0.000 claims description 2
- 235000019685 rice crackers Nutrition 0.000 claims 2
- AWOGQCSIVCQXBT-LATRNWQMSA-N (+)-Fargesin Natural products C1=C(OC)C(OC)=CC=C1[C@H]1[C@@H](CO[C@@H]2C=3C=C4OCOC4=CC=3)[C@@H]2CO1 AWOGQCSIVCQXBT-LATRNWQMSA-N 0.000 abstract description 86
- AWOGQCSIVCQXBT-LYDRAKHJSA-N Fargesin Chemical compound C1=C(OC)C(OC)=CC=C1[C@@H]1[C@H](CO[C@H]2C=3C=C4OCOC4=CC=3)[C@H]2CO1 AWOGQCSIVCQXBT-LYDRAKHJSA-N 0.000 abstract description 82
- AWOGQCSIVCQXBT-UHFFFAOYSA-N methyl piperitol Natural products C1=C(OC)C(OC)=CC=C1C1C(COC2C=3C=C4OCOC4=CC=3)C2CO1 AWOGQCSIVCQXBT-UHFFFAOYSA-N 0.000 abstract description 82
- 230000000694 effects Effects 0.000 abstract description 73
- 230000037182 bone density Effects 0.000 abstract description 69
- 208000001132 Osteoporosis Diseases 0.000 abstract description 30
- 208000020084 Bone disease Diseases 0.000 abstract description 13
- 230000008468 bone growth Effects 0.000 abstract description 5
- 230000003449 preventive effect Effects 0.000 abstract description 2
- 150000004677 hydrates Chemical class 0.000 abstract 1
- 230000001225 therapeutic effect Effects 0.000 abstract 1
- 239000002023 wood Substances 0.000 abstract 1
- 210000000988 bone and bone Anatomy 0.000 description 167
- OKKJLVBELUTLKV-UHFFFAOYSA-N Methanol Chemical compound OC OKKJLVBELUTLKV-UHFFFAOYSA-N 0.000 description 51
- 210000004027 cell Anatomy 0.000 description 51
- 238000012360 testing method Methods 0.000 description 50
- 230000001054 cortical effect Effects 0.000 description 44
- XEKOWRVHYACXOJ-UHFFFAOYSA-N Ethyl acetate Chemical compound CCOC(C)=O XEKOWRVHYACXOJ-UHFFFAOYSA-N 0.000 description 36
- 229910052500 inorganic mineral Inorganic materials 0.000 description 34
- 239000011707 mineral Substances 0.000 description 34
- 235000010755 mineral Nutrition 0.000 description 34
- 241000699670 Mus sp. Species 0.000 description 32
- 239000000243 solution Substances 0.000 description 32
- IAZDPXIOMUYVGZ-UHFFFAOYSA-N Dimethylsulphoxide Chemical compound CS(C)=O IAZDPXIOMUYVGZ-UHFFFAOYSA-N 0.000 description 31
- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 description 31
- 102000002260 Alkaline Phosphatase Human genes 0.000 description 29
- 108020004774 Alkaline Phosphatase Proteins 0.000 description 29
- VOXZDWNPVJITMN-ZBRFXRBCSA-N 17β-estradiol Chemical compound OC1=CC=C2[C@H]3CC[C@](C)([C@H](CC4)O)[C@@H]4[C@@H]3CCC2=C1 VOXZDWNPVJITMN-ZBRFXRBCSA-N 0.000 description 22
- 241000699666 Mus <mouse, genus> Species 0.000 description 22
- VLKZOEOYAKHREP-UHFFFAOYSA-N n-Hexane Chemical compound CCCCCC VLKZOEOYAKHREP-UHFFFAOYSA-N 0.000 description 21
- 239000013642 negative control Substances 0.000 description 21
- 210000002997 osteoclast Anatomy 0.000 description 21
- 238000005259 measurement Methods 0.000 description 20
- 230000003247 decreasing effect Effects 0.000 description 18
- 238000000605 extraction Methods 0.000 description 16
- 238000002360 preparation method Methods 0.000 description 16
- LFQSCWFLJHTTHZ-UHFFFAOYSA-N Ethanol Chemical compound CCO LFQSCWFLJHTTHZ-UHFFFAOYSA-N 0.000 description 15
- 102000007591 Tartrate-Resistant Acid Phosphatase Human genes 0.000 description 15
- 108010032050 Tartrate-Resistant Acid Phosphatase Proteins 0.000 description 15
- 230000037118 bone strength Effects 0.000 description 15
- 238000000034 method Methods 0.000 description 15
- 241001465754 Metazoa Species 0.000 description 14
- 210000000689 upper leg Anatomy 0.000 description 14
- 239000002609 medium Substances 0.000 description 13
- 208000035896 Twin-reversed arterial perfusion sequence Diseases 0.000 description 12
- 229940093499 ethyl acetate Drugs 0.000 description 12
- 235000019439 ethyl acetate Nutrition 0.000 description 12
- 208000010392 Bone Fractures Diseases 0.000 description 11
- 239000003795 chemical substances by application Substances 0.000 description 11
- 229960005309 estradiol Drugs 0.000 description 11
- 239000002032 methanolic fraction Substances 0.000 description 11
- 238000002156 mixing Methods 0.000 description 11
- 210000000963 osteoblast Anatomy 0.000 description 11
- 239000002904 solvent Substances 0.000 description 11
- 208000006386 Bone Resorption Diseases 0.000 description 10
- 230000024279 bone resorption Effects 0.000 description 10
- 239000003826 tablet Substances 0.000 description 10
- 238000011282 treatment Methods 0.000 description 10
- RTZKZFJDLAIYFH-UHFFFAOYSA-N Diethyl ether Chemical compound CCOCC RTZKZFJDLAIYFH-UHFFFAOYSA-N 0.000 description 9
- 239000008187 granular material Substances 0.000 description 9
- 239000000843 powder Substances 0.000 description 9
- 208000029725 Metabolic bone disease Diseases 0.000 description 8
- 230000037396 body weight Effects 0.000 description 8
- 235000013305 food Nutrition 0.000 description 8
- 239000006188 syrup Substances 0.000 description 8
- 235000020357 syrup Nutrition 0.000 description 8
- 108010010803 Gelatin Proteins 0.000 description 7
- VYPSYNLAJGMNEJ-UHFFFAOYSA-N Silicium dioxide Chemical compound O=[Si]=O VYPSYNLAJGMNEJ-UHFFFAOYSA-N 0.000 description 7
- 239000002775 capsule Substances 0.000 description 7
- 238000006243 chemical reaction Methods 0.000 description 7
- 239000008273 gelatin Substances 0.000 description 7
- 229920000159 gelatin Polymers 0.000 description 7
- 235000019322 gelatine Nutrition 0.000 description 7
- 235000011852 gelatine desserts Nutrition 0.000 description 7
- 210000004349 growth plate Anatomy 0.000 description 7
- 239000012074 organic phase Substances 0.000 description 7
- 235000000346 sugar Nutrition 0.000 description 7
- CIWBSHSKHKDKBQ-JLAZNSOCSA-N Ascorbic acid Chemical compound OC[C@H](O)[C@H]1OC(=O)C(O)=C1O CIWBSHSKHKDKBQ-JLAZNSOCSA-N 0.000 description 6
- PEDCQBHIVMGVHV-UHFFFAOYSA-N Glycerine Chemical compound OCC(O)CO PEDCQBHIVMGVHV-UHFFFAOYSA-N 0.000 description 6
- 208000030136 Marchiafava-Bignami Disease Diseases 0.000 description 6
- 108090000445 Parathyroid hormone Proteins 0.000 description 6
- 102000014128 RANK Ligand Human genes 0.000 description 6
- 108010025832 RANK Ligand Proteins 0.000 description 6
- HEMHJVSKTPXQMS-UHFFFAOYSA-M Sodium hydroxide Chemical compound [OH-].[Na+] HEMHJVSKTPXQMS-UHFFFAOYSA-M 0.000 description 6
- 208000013201 Stress fracture Diseases 0.000 description 6
- 238000007796 conventional method Methods 0.000 description 6
- 210000002745 epiphysis Anatomy 0.000 description 6
- VTYYLEPIZMXCLO-UHFFFAOYSA-L Calcium carbonate Chemical compound [Ca+2].[O-]C([O-])=O VTYYLEPIZMXCLO-UHFFFAOYSA-L 0.000 description 5
- DHCLVCXQIBBOPH-UHFFFAOYSA-N Glycerol 2-phosphate Chemical compound OCC(CO)OP(O)(O)=O DHCLVCXQIBBOPH-UHFFFAOYSA-N 0.000 description 5
- 238000005481 NMR spectroscopy Methods 0.000 description 5
- 229920002472 Starch Polymers 0.000 description 5
- 210000004369 blood Anatomy 0.000 description 5
- 239000008280 blood Substances 0.000 description 5
- 230000003833 cell viability Effects 0.000 description 5
- 235000010980 cellulose Nutrition 0.000 description 5
- 229920002678 cellulose Polymers 0.000 description 5
- 239000001913 cellulose Substances 0.000 description 5
- 230000008859 change Effects 0.000 description 5
- 230000004069 differentiation Effects 0.000 description 5
- 239000012153 distilled water Substances 0.000 description 5
- 230000012010 growth Effects 0.000 description 5
- 210000003205 muscle Anatomy 0.000 description 5
- -1 octadecyl silica Chemical compound 0.000 description 5
- 239000002244 precipitate Substances 0.000 description 5
- 239000008107 starch Substances 0.000 description 5
- 235000019698 starch Nutrition 0.000 description 5
- 210000001519 tissue Anatomy 0.000 description 5
- OYPRJOBELJOOCE-UHFFFAOYSA-N Calcium Chemical compound [Ca] OYPRJOBELJOOCE-UHFFFAOYSA-N 0.000 description 4
- WQZGKKKJIJFFOK-GASJEMHNSA-N Glucose Natural products OC[C@H]1OC(O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-GASJEMHNSA-N 0.000 description 4
- GUBGYTABKSRVRQ-QKKXKWKRSA-N Lactose Natural products OC[C@H]1O[C@@H](O[C@H]2[C@H](O)[C@@H](O)C(O)O[C@@H]2CO)[C@H](O)[C@@H](O)[C@H]1O GUBGYTABKSRVRQ-QKKXKWKRSA-N 0.000 description 4
- TWRXJAOTZQYOKJ-UHFFFAOYSA-L Magnesium chloride Chemical compound [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 4
- 206010028980 Neoplasm Diseases 0.000 description 4
- UIIMBOGNXHQVGW-UHFFFAOYSA-M Sodium bicarbonate Chemical compound [Na+].OC([O-])=O UIIMBOGNXHQVGW-UHFFFAOYSA-M 0.000 description 4
- 229930006000 Sucrose Natural products 0.000 description 4
- 238000002835 absorbance Methods 0.000 description 4
- 210000000593 adipose tissue white Anatomy 0.000 description 4
- 125000003118 aryl group Chemical group 0.000 description 4
- 230000004097 bone metabolism Effects 0.000 description 4
- DEGAKNSWVGKMLS-UHFFFAOYSA-N calcein Chemical compound O1C(=O)C2=CC=CC=C2C21C1=CC(CN(CC(O)=O)CC(O)=O)=C(O)C=C1OC1=C2C=C(CN(CC(O)=O)CC(=O)O)C(O)=C1 DEGAKNSWVGKMLS-UHFFFAOYSA-N 0.000 description 4
- 230000002308 calcification Effects 0.000 description 4
- 239000011575 calcium Substances 0.000 description 4
- 229910052791 calcium Inorganic materials 0.000 description 4
- 201000011510 cancer Diseases 0.000 description 4
- 230000008021 deposition Effects 0.000 description 4
- 239000012156 elution solvent Substances 0.000 description 4
- 238000002474 experimental method Methods 0.000 description 4
- 238000001914 filtration Methods 0.000 description 4
- 238000009472 formulation Methods 0.000 description 4
- ZTYYDUBWJTUMHW-UHFFFAOYSA-N furo[3,2-b]furan Chemical compound O1C=CC2=C1C=CO2 ZTYYDUBWJTUMHW-UHFFFAOYSA-N 0.000 description 4
- 239000000499 gel Substances 0.000 description 4
- 239000008103 glucose Substances 0.000 description 4
- 238000002347 injection Methods 0.000 description 4
- 239000007924 injection Substances 0.000 description 4
- 239000008101 lactose Substances 0.000 description 4
- HQKMJHAJHXVSDF-UHFFFAOYSA-L magnesium stearate Chemical compound [Mg+2].CCCCCCCCCCCCCCCCCC([O-])=O.CCCCCCCCCCCCCCCCCC([O-])=O HQKMJHAJHXVSDF-UHFFFAOYSA-L 0.000 description 4
- 210000001872 metatarsal bone Anatomy 0.000 description 4
- 229960002378 oftasceine Drugs 0.000 description 4
- 238000009806 oophorectomy Methods 0.000 description 4
- 239000012071 phase Substances 0.000 description 4
- 239000013641 positive control Substances 0.000 description 4
- 239000008213 purified water Substances 0.000 description 4
- 238000011160 research Methods 0.000 description 4
- 206010039073 rheumatoid arthritis Diseases 0.000 description 4
- 239000000741 silica gel Substances 0.000 description 4
- 229910002027 silica gel Inorganic materials 0.000 description 4
- 239000007901 soft capsule Substances 0.000 description 4
- 239000012192 staining solution Substances 0.000 description 4
- 239000000758 substrate Substances 0.000 description 4
- 239000005720 sucrose Substances 0.000 description 4
- CSCPPACGZOOCGX-UHFFFAOYSA-N Acetone Chemical compound CC(C)=O CSCPPACGZOOCGX-UHFFFAOYSA-N 0.000 description 3
- WEVYAHXRMPXWCK-UHFFFAOYSA-N Acetonitrile Chemical compound CC#N WEVYAHXRMPXWCK-UHFFFAOYSA-N 0.000 description 3
- 206010002091 Anaesthesia Diseases 0.000 description 3
- 108091003079 Bovine Serum Albumin Proteins 0.000 description 3
- 101000957724 Catostomus commersonii Corticoliberin-1 Proteins 0.000 description 3
- 229920002261 Corn starch Polymers 0.000 description 3
- 244000068988 Glycine max Species 0.000 description 3
- ZMXDDKWLCZADIW-UHFFFAOYSA-N N,N-Dimethylformamide Chemical compound CN(C)C=O ZMXDDKWLCZADIW-UHFFFAOYSA-N 0.000 description 3
- 235000006089 Phaseolus angularis Nutrition 0.000 description 3
- CZMRCDWAGMRECN-UGDNZRGBSA-N Sucrose Chemical compound O[C@H]1[C@H](O)[C@@H](CO)O[C@@]1(CO)O[C@@H]1[C@H](O)[C@@H](O)[C@H](O)[C@@H](CO)O1 CZMRCDWAGMRECN-UGDNZRGBSA-N 0.000 description 3
- 240000007098 Vigna angularis Species 0.000 description 3
- 235000010711 Vigna angularis Nutrition 0.000 description 3
- 0 [1*]C1=CC([C@@H]2OC[C@]3([H])[C@@H](C4=CC([4*])=C5OCOC5=C4)OC[C@]23[H])=CC(O[3*])=C1O[2*] Chemical compound [1*]C1=CC([C@@H]2OC[C@]3([H])[C@@H](C4=CC([4*])=C5OCOC5=C4)OC[C@]23[H])=CC(O[3*])=C1O[2*] 0.000 description 3
- 210000003486 adipose tissue brown Anatomy 0.000 description 3
- 230000037005 anaesthesia Effects 0.000 description 3
- 239000007864 aqueous solution Substances 0.000 description 3
- 206010003246 arthritis Diseases 0.000 description 3
- 229960005070 ascorbic acid Drugs 0.000 description 3
- 239000007640 basal medium Substances 0.000 description 3
- 208000016738 bone Paget disease Diseases 0.000 description 3
- 229940112822 chewing gum Drugs 0.000 description 3
- 235000015218 chewing gum Nutrition 0.000 description 3
- KRKNYBCHXYNGOX-UHFFFAOYSA-N citric acid Chemical compound OC(=O)CC(O)(C(O)=O)CC(O)=O KRKNYBCHXYNGOX-UHFFFAOYSA-N 0.000 description 3
- 239000012881 co-culture medium Substances 0.000 description 3
- 239000011248 coating agent Substances 0.000 description 3
- 238000004440 column chromatography Methods 0.000 description 3
- 239000008120 corn starch Substances 0.000 description 3
- 239000008367 deionised water Substances 0.000 description 3
- 229910021641 deionized water Inorganic materials 0.000 description 3
- 229960004132 diethyl ether Drugs 0.000 description 3
- 239000003651 drinking water Substances 0.000 description 3
- 235000020188 drinking water Nutrition 0.000 description 3
- 239000012149 elution buffer Substances 0.000 description 3
- 239000003995 emulsifying agent Substances 0.000 description 3
- 238000005516 engineering process Methods 0.000 description 3
- 239000012091 fetal bovine serum Substances 0.000 description 3
- 210000003754 fetus Anatomy 0.000 description 3
- 235000011187 glycerol Nutrition 0.000 description 3
- 239000001963 growth medium Substances 0.000 description 3
- 239000007902 hard capsule Substances 0.000 description 3
- 239000001863 hydroxypropyl cellulose Substances 0.000 description 3
- 235000010977 hydroxypropyl cellulose Nutrition 0.000 description 3
- 229940071676 hydroxypropylcellulose Drugs 0.000 description 3
- 230000006698 induction Effects 0.000 description 3
- 210000003141 lower extremity Anatomy 0.000 description 3
- 230000036210 malignancy Effects 0.000 description 3
- 239000012528 membrane Substances 0.000 description 3
- 239000003960 organic solvent Substances 0.000 description 3
- 208000005368 osteomalacia Diseases 0.000 description 3
- 238000011321 prophylaxis Methods 0.000 description 3
- 238000000746 purification Methods 0.000 description 3
- 230000002829 reductive effect Effects 0.000 description 3
- 210000002966 serum Anatomy 0.000 description 3
- 239000007787 solid Substances 0.000 description 3
- 238000010186 staining Methods 0.000 description 3
- 238000012353 t test Methods 0.000 description 3
- 210000002303 tibia Anatomy 0.000 description 3
- 210000004291 uterus Anatomy 0.000 description 3
- 239000011647 vitamin D3 Substances 0.000 description 3
- QYSXJUFSXHHAJI-YRZJJWOYSA-N vitamin D3 Chemical compound C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C\C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-YRZJJWOYSA-N 0.000 description 3
- 238000005160 1H NMR spectroscopy Methods 0.000 description 2
- QKNYBSVHEMOAJP-UHFFFAOYSA-N 2-amino-2-(hydroxymethyl)propane-1,3-diol;hydron;chloride Chemical compound Cl.OCC(N)(CO)CO QKNYBSVHEMOAJP-UHFFFAOYSA-N 0.000 description 2
- 229920001817 Agar Polymers 0.000 description 2
- 206010006187 Breast cancer Diseases 0.000 description 2
- 208000026310 Breast neoplasm Diseases 0.000 description 2
- 102000055006 Calcitonin Human genes 0.000 description 2
- 108060001064 Calcitonin Proteins 0.000 description 2
- FBPFZTCFMRRESA-KVTDHHQDSA-N D-Mannitol Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-KVTDHHQDSA-N 0.000 description 2
- 239000001856 Ethyl cellulose Substances 0.000 description 2
- ZZSNKZQZMQGXPY-UHFFFAOYSA-N Ethyl cellulose Chemical compound CCOCC1OC(OC)C(OCC)C(OCC)C1OC1C(O)C(O)C(OC)C(CO)O1 ZZSNKZQZMQGXPY-UHFFFAOYSA-N 0.000 description 2
- WSFSSNUMVMOOMR-UHFFFAOYSA-N Formaldehyde Chemical compound O=C WSFSSNUMVMOOMR-UHFFFAOYSA-N 0.000 description 2
- 229920002153 Hydroxypropyl cellulose Polymers 0.000 description 2
- 208000037147 Hypercalcaemia Diseases 0.000 description 2
- 239000002211 L-ascorbic acid Substances 0.000 description 2
- 235000000069 L-ascorbic acid Nutrition 0.000 description 2
- 229930195725 Mannitol Natural products 0.000 description 2
- 206010027476 Metastases Diseases 0.000 description 2
- LRHPLDYGYMQRHN-UHFFFAOYSA-N N-Butanol Chemical compound CCCCO LRHPLDYGYMQRHN-UHFFFAOYSA-N 0.000 description 2
- 206010049088 Osteopenia Diseases 0.000 description 2
- QGMRQYFBGABWDR-UHFFFAOYSA-M Pentobarbital sodium Chemical compound [Na+].CCCC(C)C1(CC)C(=O)NC(=O)[N-]C1=O QGMRQYFBGABWDR-UHFFFAOYSA-M 0.000 description 2
- 101150107869 Sarg gene Proteins 0.000 description 2
- 244000269722 Thea sinensis Species 0.000 description 2
- 235000005811 Viola adunca Nutrition 0.000 description 2
- 240000009038 Viola odorata Species 0.000 description 2
- 235000013487 Viola odorata Nutrition 0.000 description 2
- 235000002254 Viola papilionacea Nutrition 0.000 description 2
- IOMLBTHPCVDRHM-UHFFFAOYSA-N [3-[(2,4-dimethylphenyl)carbamoyl]naphthalen-2-yl] dihydrogen phosphate Chemical compound CC1=CC(C)=CC=C1NC(=O)C1=CC2=CC=CC=C2C=C1OP(O)(O)=O IOMLBTHPCVDRHM-UHFFFAOYSA-N 0.000 description 2
- 230000004913 activation Effects 0.000 description 2
- 239000008272 agar Substances 0.000 description 2
- 125000001931 aliphatic group Chemical group 0.000 description 2
- 238000004458 analytical method Methods 0.000 description 2
- 239000008346 aqueous phase Substances 0.000 description 2
- 230000015572 biosynthetic process Effects 0.000 description 2
- 230000018678 bone mineralization Effects 0.000 description 2
- 229960004015 calcitonin Drugs 0.000 description 2
- BBBFJLBPOGFECG-VJVYQDLKSA-N calcitonin Chemical compound N([C@H](C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC=1NC=NC=1)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC=1C=CC(O)=CC=1)C(=O)N1[C@@H](CCC1)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H]([C@@H](C)O)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H]([C@@H](C)O)C(=O)NCC(=O)N[C@@H](CO)C(=O)NCC(=O)N[C@@H]([C@@H](C)O)C(=O)N1[C@@H](CCC1)C(N)=O)C(C)C)C(=O)[C@@H]1CSSC[C@H](N)C(=O)N[C@@H](CO)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CO)C(=O)N[C@@H]([C@@H](C)O)C(=O)N1 BBBFJLBPOGFECG-VJVYQDLKSA-N 0.000 description 2
- 229910000019 calcium carbonate Inorganic materials 0.000 description 2
- 235000010216 calcium carbonate Nutrition 0.000 description 2
- OSGAYBCDTDRGGQ-UHFFFAOYSA-L calcium sulfate Chemical compound [Ca+2].[O-]S([O-])(=O)=O OSGAYBCDTDRGGQ-UHFFFAOYSA-L 0.000 description 2
- 238000001460 carbon-13 nuclear magnetic resonance spectrum Methods 0.000 description 2
- 239000001768 carboxy methyl cellulose Substances 0.000 description 2
- 239000003153 chemical reaction reagent Substances 0.000 description 2
- 238000000576 coating method Methods 0.000 description 2
- 239000003086 colorant Substances 0.000 description 2
- 230000000295 complement effect Effects 0.000 description 2
- 239000003636 conditioned culture medium Substances 0.000 description 2
- 235000014510 cooky Nutrition 0.000 description 2
- 238000011161 development Methods 0.000 description 2
- 230000018109 developmental process Effects 0.000 description 2
- 201000010099 disease Diseases 0.000 description 2
- 208000037265 diseases, disorders, signs and symptoms Diseases 0.000 description 2
- 239000008298 dragée Substances 0.000 description 2
- 239000003814 drug Substances 0.000 description 2
- 235000013399 edible fruits Nutrition 0.000 description 2
- 238000010828 elution Methods 0.000 description 2
- 229940011871 estrogen Drugs 0.000 description 2
- 239000000262 estrogen Substances 0.000 description 2
- 235000019325 ethyl cellulose Nutrition 0.000 description 2
- 229920001249 ethyl cellulose Polymers 0.000 description 2
- 238000011156 evaluation Methods 0.000 description 2
- 235000021050 feed intake Nutrition 0.000 description 2
- 239000000706 filtrate Substances 0.000 description 2
- 235000011194 food seasoning agent Nutrition 0.000 description 2
- 239000012737 fresh medium Substances 0.000 description 2
- 235000011389 fruit/vegetable juice Nutrition 0.000 description 2
- 230000006870 function Effects 0.000 description 2
- 238000004128 high performance liquid chromatography Methods 0.000 description 2
- 230000000148 hypercalcaemia Effects 0.000 description 2
- 208000030915 hypercalcemia disease Diseases 0.000 description 2
- 230000002757 inflammatory effect Effects 0.000 description 2
- 230000005764 inhibitory process Effects 0.000 description 2
- 238000007912 intraperitoneal administration Methods 0.000 description 2
- 235000015110 jellies Nutrition 0.000 description 2
- 239000008274 jelly Substances 0.000 description 2
- 229920005610 lignin Polymers 0.000 description 2
- 210000004185 liver Anatomy 0.000 description 2
- 238000011068 loading method Methods 0.000 description 2
- 239000000314 lubricant Substances 0.000 description 2
- 210000002540 macrophage Anatomy 0.000 description 2
- 229910001629 magnesium chloride Inorganic materials 0.000 description 2
- 235000019359 magnesium stearate Nutrition 0.000 description 2
- 239000000594 mannitol Substances 0.000 description 2
- 235000010355 mannitol Nutrition 0.000 description 2
- 238000004519 manufacturing process Methods 0.000 description 2
- 239000003550 marker Substances 0.000 description 2
- 230000009401 metastasis Effects 0.000 description 2
- 208000030212 nutrition disease Diseases 0.000 description 2
- 230000003287 optical effect Effects 0.000 description 2
- WEXRUCMBJFQVBZ-UHFFFAOYSA-N pentobarbital Chemical compound CCCC(C)C1(CC)C(=O)NC(=O)NC1=O WEXRUCMBJFQVBZ-UHFFFAOYSA-N 0.000 description 2
- 239000000546 pharmaceutical excipient Substances 0.000 description 2
- 239000001267 polyvinylpyrrolidone Substances 0.000 description 2
- 229920000036 polyvinylpyrrolidone Polymers 0.000 description 2
- 235000013855 polyvinylpyrrolidone Nutrition 0.000 description 2
- 239000002243 precursor Substances 0.000 description 2
- 238000004237 preparative chromatography Methods 0.000 description 2
- 230000002265 prevention Effects 0.000 description 2
- 239000000047 product Substances 0.000 description 2
- 230000002441 reversible effect Effects 0.000 description 2
- CVHZOJJKTDOEJC-UHFFFAOYSA-N saccharin Chemical compound C1=CC=C2C(=O)NS(=O)(=O)C2=C1 CVHZOJJKTDOEJC-UHFFFAOYSA-N 0.000 description 2
- 229940081974 saccharin Drugs 0.000 description 2
- 235000019204 saccharin Nutrition 0.000 description 2
- 239000000901 saccharin and its Na,K and Ca salt Substances 0.000 description 2
- 239000000377 silicon dioxide Substances 0.000 description 2
- 235000020183 skimmed milk Nutrition 0.000 description 2
- HELHAJAZNSDZJO-OLXYHTOASA-L sodium L-tartrate Chemical compound [Na+].[Na+].[O-]C(=O)[C@H](O)[C@@H](O)C([O-])=O HELHAJAZNSDZJO-OLXYHTOASA-L 0.000 description 2
- 229910000030 sodium bicarbonate Inorganic materials 0.000 description 2
- 235000017557 sodium bicarbonate Nutrition 0.000 description 2
- 239000001433 sodium tartrate Substances 0.000 description 2
- 229960002167 sodium tartrate Drugs 0.000 description 2
- 235000011004 sodium tartrates Nutrition 0.000 description 2
- 235000014214 soft drink Nutrition 0.000 description 2
- 238000001228 spectrum Methods 0.000 description 2
- 210000000952 spleen Anatomy 0.000 description 2
- 239000003381 stabilizer Substances 0.000 description 2
- 230000000638 stimulation Effects 0.000 description 2
- UCSJYZPVAKXKNQ-HZYVHMACSA-N streptomycin Chemical compound CN[C@H]1[C@H](O)[C@@H](O)[C@H](CO)O[C@H]1O[C@@H]1[C@](C=O)(O)[C@H](C)O[C@H]1O[C@@H]1[C@@H](NC(N)=N)[C@H](O)[C@@H](NC(N)=N)[C@H](O)[C@H]1O UCSJYZPVAKXKNQ-HZYVHMACSA-N 0.000 description 2
- 239000007940 sugar coated tablet Substances 0.000 description 2
- 235000013616 tea Nutrition 0.000 description 2
- OGBMKVWORPGQRR-UMXFMPSGSA-N teriparatide Chemical compound C([C@H](NC(=O)[C@H](CCSC)NC(=O)[C@H](CC(C)C)NC(=O)[C@H](CCC(N)=O)NC(=O)[C@@H](NC(=O)[C@H](CCC(O)=O)NC(=O)[C@H](CO)NC(=O)[C@@H](NC(=O)[C@@H](N)CO)C(C)C)[C@@H](C)CC)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC(C)C)C(=O)NCC(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CO)C(=O)N[C@@H](CCSC)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CCC(O)=O)C(=O)N[C@@H](CC=1C2=CC=CC=C2NC=1)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCCNC(N)=N)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CCCCN)C(=O)N[C@@H](CC(C)C)C(=O)N[C@@H](CCC(N)=O)C(=O)N[C@@H](CC(O)=O)C(=O)N[C@@H](C(C)C)C(=O)N[C@@H](CC=1N=CNC=1)C(=O)N[C@@H](CC(N)=O)C(=O)N[C@@H](CC=1C=CC=CC=1)C(O)=O)C1=CNC=N1 OGBMKVWORPGQRR-UMXFMPSGSA-N 0.000 description 2
- 238000004809 thin layer chromatography Methods 0.000 description 2
- 230000000472 traumatic effect Effects 0.000 description 2
- PJVXUVWGSCCGHT-ZPYZYFCMSA-N (2r,3s,4r,5r)-2,3,4,5,6-pentahydroxyhexanal;(3s,4r,5r)-1,3,4,5,6-pentahydroxyhexan-2-one Chemical compound OC[C@@H](O)[C@@H](O)[C@H](O)[C@@H](O)C=O.OC[C@@H](O)[C@@H](O)[C@H](O)C(=O)CO PJVXUVWGSCCGHT-ZPYZYFCMSA-N 0.000 description 1
- IXPNQXFRVYWDDI-UHFFFAOYSA-N 1-methyl-2,4-dioxo-1,3-diazinane-5-carboximidamide Chemical compound CN1CC(C(N)=N)C(=O)NC1=O IXPNQXFRVYWDDI-UHFFFAOYSA-N 0.000 description 1
- 238000001644 13C nuclear magnetic resonance spectroscopy Methods 0.000 description 1
- AZKSAVLVSZKNRD-UHFFFAOYSA-M 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Chemical compound [Br-].S1C(C)=C(C)N=C1[N+]1=NC(C=2C=CC=CC=2)=NN1C1=CC=CC=C1 AZKSAVLVSZKNRD-UHFFFAOYSA-M 0.000 description 1
- SARKXLKWFKNUMR-UHFFFAOYSA-N 4-benzamido-5-chloro-2-methylbenzenediazonium;chloride Chemical compound [Cl-].C1=C([N+]#N)C(C)=CC(NC(=O)C=2C=CC=CC=2)=C1Cl SARKXLKWFKNUMR-UHFFFAOYSA-N 0.000 description 1
- QTBSBXVTEAMEQO-UHFFFAOYSA-M Acetate Chemical compound CC([O-])=O QTBSBXVTEAMEQO-UHFFFAOYSA-M 0.000 description 1
- GUBGYTABKSRVRQ-XLOQQCSPSA-N Alpha-Lactose Chemical compound O[C@@H]1[C@@H](O)[C@@H](O)[C@@H](CO)O[C@H]1O[C@@H]1[C@@H](CO)O[C@H](O)[C@H](O)[C@H]1O GUBGYTABKSRVRQ-XLOQQCSPSA-N 0.000 description 1
- 239000000592 Artificial Cell Substances 0.000 description 1
- 206010003694 Atrophy Diseases 0.000 description 1
- 206010065687 Bone loss Diseases 0.000 description 1
- 101100502395 Caenorhabditis elegans far-2 gene Proteins 0.000 description 1
- 229920002134 Carboxymethyl cellulose Polymers 0.000 description 1
- 241000182341 Cubitermes group Species 0.000 description 1
- 229920000858 Cyclodextrin Polymers 0.000 description 1
- 102000004127 Cytokines Human genes 0.000 description 1
- 108090000695 Cytokines Proteins 0.000 description 1
- FBPFZTCFMRRESA-FSIIMWSLSA-N D-Glucitol Natural products OC[C@H](O)[C@H](O)[C@@H](O)[C@H](O)CO FBPFZTCFMRRESA-FSIIMWSLSA-N 0.000 description 1
- FBPFZTCFMRRESA-JGWLITMVSA-N D-glucitol Chemical compound OC[C@H](O)[C@@H](O)[C@H](O)[C@H](O)CO FBPFZTCFMRRESA-JGWLITMVSA-N 0.000 description 1
- 229920001353 Dextrin Polymers 0.000 description 1
- 239000004375 Dextrin Substances 0.000 description 1
- 239000004278 EU approved seasoning Substances 0.000 description 1
- LYCAIKOWRPUZTN-UHFFFAOYSA-N Ethylene glycol Chemical compound OCCO LYCAIKOWRPUZTN-UHFFFAOYSA-N 0.000 description 1
- 241001071795 Gentiana Species 0.000 description 1
- 241000202807 Glycyrrhiza Species 0.000 description 1
- 101001135770 Homo sapiens Parathyroid hormone Proteins 0.000 description 1
- 101001135995 Homo sapiens Probable peptidyl-tRNA hydrolase Proteins 0.000 description 1
- 241000186660 Lactobacillus Species 0.000 description 1
- 238000000134 MTT assay Methods 0.000 description 1
- 231100000002 MTT assay Toxicity 0.000 description 1
- 241000218378 Magnolia Species 0.000 description 1
- CERQOIWHTDAKMF-UHFFFAOYSA-M Methacrylate Chemical compound CC(=C)C([O-])=O CERQOIWHTDAKMF-UHFFFAOYSA-M 0.000 description 1
- VVQNEPGJFQJSBK-UHFFFAOYSA-N Methyl methacrylate Chemical compound COC(=O)C(C)=C VVQNEPGJFQJSBK-UHFFFAOYSA-N 0.000 description 1
- 229920000168 Microcrystalline cellulose Polymers 0.000 description 1
- WHNWPMSKXPGLAX-UHFFFAOYSA-N N-Vinyl-2-pyrrolidone Chemical compound C=CN1CCCC1=O WHNWPMSKXPGLAX-UHFFFAOYSA-N 0.000 description 1
- 239000004677 Nylon Substances 0.000 description 1
- LXAATRGTGHYGJH-UHFFFAOYSA-N OP(O)(O)=O.[O-][N+](=O)C1=CC=C([Na])C=C1 Chemical compound OP(O)(O)=O.[O-][N+](=O)C1=CC=C([Na])C=C1 LXAATRGTGHYGJH-UHFFFAOYSA-N 0.000 description 1
- 102000004067 Osteocalcin Human genes 0.000 description 1
- 108090000573 Osteocalcin Proteins 0.000 description 1
- 206010034156 Pathological fracture Diseases 0.000 description 1
- 229930182555 Penicillin Natural products 0.000 description 1
- JGSARLDLIJGVTE-MBNYWOFBSA-N Penicillin G Chemical compound N([C@H]1[C@H]2SC([C@@H](N2C1=O)C(O)=O)(C)C)C(=O)CC1=CC=CC=C1 JGSARLDLIJGVTE-MBNYWOFBSA-N 0.000 description 1
- 208000006735 Periostitis Diseases 0.000 description 1
- 241000533293 Sesbania emerus Species 0.000 description 1
- 229920001800 Shellac Polymers 0.000 description 1
- UIIMBOGNXHQVGW-DEQYMQKBSA-M Sodium bicarbonate-14C Chemical compound [Na+].O[14C]([O-])=O UIIMBOGNXHQVGW-DEQYMQKBSA-M 0.000 description 1
- 208000005250 Spontaneous Fractures Diseases 0.000 description 1
- 108010049264 Teriparatide Proteins 0.000 description 1
- HEDRZPFGACZZDS-MICDWDOJSA-N Trichloro(2H)methane Chemical class [2H]C(Cl)(Cl)Cl HEDRZPFGACZZDS-MICDWDOJSA-N 0.000 description 1
- 229930003316 Vitamin D Natural products 0.000 description 1
- QYSXJUFSXHHAJI-XFEUOLMDSA-N Vitamin D3 Natural products C1(/[C@@H]2CC[C@@H]([C@]2(CCC1)C)[C@H](C)CCCC(C)C)=C/C=C1\C[C@@H](O)CCC1=C QYSXJUFSXHHAJI-XFEUOLMDSA-N 0.000 description 1
- 208000021017 Weight Gain Diseases 0.000 description 1
- 235000010724 Wisteria floribunda Nutrition 0.000 description 1
- YCGKWKHHSRGNSK-QPAOKJHLSA-N [H][C@]12CO[C@@H](C3=CC(OC)=C(OC)C=C3)[C@@]1([H])CO[C@@H]2C1=CC=C2CCOC2=C1 Chemical compound [H][C@]12CO[C@@H](C3=CC(OC)=C(OC)C=C3)[C@@]1([H])CO[C@@H]2C1=CC=C2CCOC2=C1 YCGKWKHHSRGNSK-QPAOKJHLSA-N 0.000 description 1
- 230000002159 abnormal effect Effects 0.000 description 1
- 238000010521 absorption reaction Methods 0.000 description 1
- 230000001133 acceleration Effects 0.000 description 1
- DPXJVFZANSGRMM-UHFFFAOYSA-N acetic acid;2,3,4,5,6-pentahydroxyhexanal;sodium Chemical compound [Na].CC(O)=O.OCC(O)C(O)C(O)C(O)C=O DPXJVFZANSGRMM-UHFFFAOYSA-N 0.000 description 1
- 230000003213 activating effect Effects 0.000 description 1
- 239000000654 additive Substances 0.000 description 1
- 239000000443 aerosol Substances 0.000 description 1
- 238000007605 air drying Methods 0.000 description 1
- RGCKGOZRHPZPFP-UHFFFAOYSA-N alizarin Chemical compound C1=CC=C2C(=O)C3=C(O)C(O)=CC=C3C(=O)C2=C1 RGCKGOZRHPZPFP-UHFFFAOYSA-N 0.000 description 1
- 230000004075 alteration Effects 0.000 description 1
- 239000000730 antalgic agent Substances 0.000 description 1
- 238000013459 approach Methods 0.000 description 1
- 235000010323 ascorbic acid Nutrition 0.000 description 1
- 239000011668 ascorbic acid Substances 0.000 description 1
- 238000003556 assay Methods 0.000 description 1
- 230000037444 atrophy Effects 0.000 description 1
- 238000005452 bending Methods 0.000 description 1
- 230000009286 beneficial effect Effects 0.000 description 1
- WQZGKKKJIJFFOK-VFUOTHLCSA-N beta-D-glucose Chemical compound OC[C@H]1O[C@@H](O)[C@H](O)[C@@H](O)[C@@H]1O WQZGKKKJIJFFOK-VFUOTHLCSA-N 0.000 description 1
- 239000011230 binding agent Substances 0.000 description 1
- 230000000740 bleeding effect Effects 0.000 description 1
- 210000002449 bone cell Anatomy 0.000 description 1
- 235000008429 bread Nutrition 0.000 description 1
- 235000021329 brown rice Nutrition 0.000 description 1
- 239000006189 buccal tablet Substances 0.000 description 1
- 239000007853 buffer solution Substances 0.000 description 1
- 239000006172 buffering agent Substances 0.000 description 1
- 235000001465 calcium Nutrition 0.000 description 1
- 229940043430 calcium compound Drugs 0.000 description 1
- 150000001674 calcium compounds Chemical class 0.000 description 1
- 230000003913 calcium metabolism Effects 0.000 description 1
- 239000001506 calcium phosphate Substances 0.000 description 1
- 229910000389 calcium phosphate Inorganic materials 0.000 description 1
- 235000011010 calcium phosphates Nutrition 0.000 description 1
- 159000000007 calcium salts Chemical class 0.000 description 1
- 239000000378 calcium silicate Substances 0.000 description 1
- 229910052918 calcium silicate Inorganic materials 0.000 description 1
- 235000011132 calcium sulphate Nutrition 0.000 description 1
- OYACROKNLOSFPA-UHFFFAOYSA-N calcium;dioxido(oxo)silane Chemical compound [Ca+2].[O-][Si]([O-])=O OYACROKNLOSFPA-UHFFFAOYSA-N 0.000 description 1
- 235000010948 carboxy methyl cellulose Nutrition 0.000 description 1
- 239000008112 carboxymethyl-cellulose Substances 0.000 description 1
- 239000000969 carrier Substances 0.000 description 1
- 210000003321 cartilage cell Anatomy 0.000 description 1
- 230000003915 cell function Effects 0.000 description 1
- 230000004663 cell proliferation Effects 0.000 description 1
- 239000006285 cell suspension Substances 0.000 description 1
- 150000003841 chloride salts Chemical class 0.000 description 1
- 230000002060 circadian Effects 0.000 description 1
- 238000003501 co-culture Methods 0.000 description 1
- 239000007931 coated granule Substances 0.000 description 1
- 238000002591 computed tomography Methods 0.000 description 1
- 239000012141 concentrate Substances 0.000 description 1
- 210000002808 connective tissue Anatomy 0.000 description 1
- 239000000356 contaminant Substances 0.000 description 1
- 229960000913 crospovidone Drugs 0.000 description 1
- 239000000287 crude extract Substances 0.000 description 1
- 230000009089 cytolysis Effects 0.000 description 1
- GVJHHUAWPYXKBD-UHFFFAOYSA-N d-alpha-tocopherol Natural products OC1=C(C)C(C)=C2OC(CCCC(C)CCCC(C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-UHFFFAOYSA-N 0.000 description 1
- 230000001419 dependent effect Effects 0.000 description 1
- 235000019425 dextrin Nutrition 0.000 description 1
- 235000005911 diet Nutrition 0.000 description 1
- 230000000378 dietary effect Effects 0.000 description 1
- 235000014113 dietary fatty acids Nutrition 0.000 description 1
- XEYBRNLFEZDVAW-ARSRFYASSA-N dinoprostone Chemical compound CCCCC[C@H](O)\C=C\[C@H]1[C@H](O)CC(=O)[C@@H]1C\C=C/CCCC(O)=O XEYBRNLFEZDVAW-ARSRFYASSA-N 0.000 description 1
- KAKKHKRHCKCAGH-UHFFFAOYSA-L disodium;(4-nitrophenyl) phosphate;hexahydrate Chemical compound O.O.O.O.O.O.[Na+].[Na+].[O-][N+](=O)C1=CC=C(OP([O-])([O-])=O)C=C1 KAKKHKRHCKCAGH-UHFFFAOYSA-L 0.000 description 1
- 238000002224 dissection Methods 0.000 description 1
- 239000012738 dissolution medium Substances 0.000 description 1
- 239000002552 dosage form Substances 0.000 description 1
- 231100000673 dose–response relationship Toxicity 0.000 description 1
- 238000012137 double-staining Methods 0.000 description 1
- 229940079593 drug Drugs 0.000 description 1
- 230000008030 elimination Effects 0.000 description 1
- 238000003379 elimination reaction Methods 0.000 description 1
- 239000000839 emulsion Substances 0.000 description 1
- 239000002662 enteric coated tablet Substances 0.000 description 1
- 230000001076 estrogenic effect Effects 0.000 description 1
- 238000009207 exercise therapy Methods 0.000 description 1
- 239000000194 fatty acid Substances 0.000 description 1
- 229930195729 fatty acid Natural products 0.000 description 1
- 239000000796 flavoring agent Substances 0.000 description 1
- 235000019634 flavors Nutrition 0.000 description 1
- 238000012757 fluorescence staining Methods 0.000 description 1
- 238000004108 freeze drying Methods 0.000 description 1
- 235000015203 fruit juice Nutrition 0.000 description 1
- 235000021552 granulated sugar Nutrition 0.000 description 1
- 210000001564 haversian system Anatomy 0.000 description 1
- 230000003862 health status Effects 0.000 description 1
- 229940125697 hormonal agent Drugs 0.000 description 1
- 229940088597 hormone Drugs 0.000 description 1
- 239000005556 hormone Substances 0.000 description 1
- 102000058004 human PTH Human genes 0.000 description 1
- 239000008172 hydrogenated vegetable oil Substances 0.000 description 1
- 239000001866 hydroxypropyl methyl cellulose Substances 0.000 description 1
- 235000010979 hydroxypropyl methyl cellulose Nutrition 0.000 description 1
- 229920003088 hydroxypropyl methyl cellulose Polymers 0.000 description 1
- UFVKGYZPFZQRLF-UHFFFAOYSA-N hydroxypropyl methyl cellulose Chemical compound OC1C(O)C(OC)OC(CO)C1OC1C(O)C(O)C(OC2C(C(O)C(OC3C(C(O)C(O)C(CO)O3)O)C(CO)O2)O)C(CO)O1 UFVKGYZPFZQRLF-UHFFFAOYSA-N 0.000 description 1
- 229920003132 hydroxypropyl methylcellulose phthalate Polymers 0.000 description 1
- 229940031704 hydroxypropyl methylcellulose phthalate Drugs 0.000 description 1
- 238000001727 in vivo Methods 0.000 description 1
- 238000011534 incubation Methods 0.000 description 1
- 239000004615 ingredient Substances 0.000 description 1
- 235000021539 instant coffee Nutrition 0.000 description 1
- 230000009545 invasion Effects 0.000 description 1
- LTINPJMVDKPJJI-UHFFFAOYSA-N iodinated glycerol Chemical compound CC(I)C1OCC(CO)O1 LTINPJMVDKPJJI-UHFFFAOYSA-N 0.000 description 1
- 210000003734 kidney Anatomy 0.000 description 1
- 229940039696 lactobacillus Drugs 0.000 description 1
- XMGQYMWWDOXHJM-UHFFFAOYSA-N limonene Chemical compound CC(=C)C1CCC(C)=CC1 XMGQYMWWDOXHJM-UHFFFAOYSA-N 0.000 description 1
- 239000007788 liquid Substances 0.000 description 1
- 238000012594 liquid chromatography nuclear magnetic resonance Methods 0.000 description 1
- 239000012669 liquid formulation Substances 0.000 description 1
- 238000000622 liquid--liquid extraction Methods 0.000 description 1
- 229960003511 macrogol Drugs 0.000 description 1
- 238000004949 mass spectrometry Methods 0.000 description 1
- 230000035800 maturation Effects 0.000 description 1
- 230000002503 metabolic effect Effects 0.000 description 1
- 229940016286 microcrystalline cellulose Drugs 0.000 description 1
- 235000019813 microcrystalline cellulose Nutrition 0.000 description 1
- 239000008108 microcrystalline cellulose Substances 0.000 description 1
- 235000013336 milk Nutrition 0.000 description 1
- 239000008267 milk Substances 0.000 description 1
- 210000004080 milk Anatomy 0.000 description 1
- 230000009456 molecular mechanism Effects 0.000 description 1
- 210000001616 monocyte Anatomy 0.000 description 1
- 150000004682 monohydrates Chemical class 0.000 description 1
- 231100000957 no side effect Toxicity 0.000 description 1
- 238000000655 nuclear magnetic resonance spectrum Methods 0.000 description 1
- 235000016709 nutrition Nutrition 0.000 description 1
- 229920001778 nylon Polymers 0.000 description 1
- 239000003921 oil Substances 0.000 description 1
- 235000019198 oils Nutrition 0.000 description 1
- 239000007968 orange flavor Substances 0.000 description 1
- 150000007524 organic acids Chemical class 0.000 description 1
- 201000008482 osteoarthritis Diseases 0.000 description 1
- 210000001672 ovary Anatomy 0.000 description 1
- 230000036961 partial effect Effects 0.000 description 1
- 229940049954 penicillin Drugs 0.000 description 1
- 229960001412 pentobarbital Drugs 0.000 description 1
- 229960002275 pentobarbital sodium Drugs 0.000 description 1
- 208000028169 periodontal disease Diseases 0.000 description 1
- 210000003460 periosteum Anatomy 0.000 description 1
- 230000002093 peripheral effect Effects 0.000 description 1
- 230000026731 phosphorylation Effects 0.000 description 1
- 238000006366 phosphorylation reaction Methods 0.000 description 1
- XNGIFLGASWRNHJ-UHFFFAOYSA-L phthalate(2-) Chemical compound [O-]C(=O)C1=CC=CC=C1C([O-])=O XNGIFLGASWRNHJ-UHFFFAOYSA-L 0.000 description 1
- 239000006187 pill Substances 0.000 description 1
- 238000013001 point bending Methods 0.000 description 1
- 229920000642 polymer Polymers 0.000 description 1
- 235000010482 polyoxyethylene sorbitan monooleate Nutrition 0.000 description 1
- 229920000053 polysorbate 80 Polymers 0.000 description 1
- 235000013809 polyvinylpolypyrrolidone Nutrition 0.000 description 1
- 229920000523 polyvinylpolypyrrolidone Polymers 0.000 description 1
- 239000011148 porous material Substances 0.000 description 1
- XAEFZNCEHLXOMS-UHFFFAOYSA-M potassium benzoate Chemical compound [K+].[O-]C(=O)C1=CC=CC=C1 XAEFZNCEHLXOMS-UHFFFAOYSA-M 0.000 description 1
- 229920001592 potato starch Polymers 0.000 description 1
- 230000035935 pregnancy Effects 0.000 description 1
- 238000012545 processing Methods 0.000 description 1
- 230000035755 proliferation Effects 0.000 description 1
- 230000000069 prophylactic effect Effects 0.000 description 1
- 102000004169 proteins and genes Human genes 0.000 description 1
- 108090000623 proteins and genes Proteins 0.000 description 1
- 238000000425 proton nuclear magnetic resonance spectrum Methods 0.000 description 1
- 238000011084 recovery Methods 0.000 description 1
- 230000009467 reduction Effects 0.000 description 1
- BOLDJAUMGUJJKM-LSDHHAIUSA-N renifolin D Natural products CC(=C)[C@@H]1Cc2c(O)c(O)ccc2[C@H]1CC(=O)c3ccc(O)cc3O BOLDJAUMGUJJKM-LSDHHAIUSA-N 0.000 description 1
- HFHDHCJBZVLPGP-UHFFFAOYSA-N schardinger α-dextrin Chemical compound O1C(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC(C(O)C2O)C(CO)OC2OC(C(C2O)O)C(CO)OC2OC2C(O)C(O)C1OC2CO HFHDHCJBZVLPGP-UHFFFAOYSA-N 0.000 description 1
- 230000028327 secretion Effects 0.000 description 1
- 230000000276 sedentary effect Effects 0.000 description 1
- 239000004208 shellac Substances 0.000 description 1
- 229940113147 shellac Drugs 0.000 description 1
- 235000013874 shellac Nutrition 0.000 description 1
- ZLGIYFNHBLSMPS-ATJNOEHPSA-N shellac Chemical compound OCCCCCC(O)C(O)CCCCCCCC(O)=O.C1C23[C@H](C(O)=O)CCC2[C@](C)(CO)[C@@H]1C(C(O)=O)=C[C@@H]3O ZLGIYFNHBLSMPS-ATJNOEHPSA-N 0.000 description 1
- 235000020374 simple syrup Nutrition 0.000 description 1
- 239000007974 sodium acetate buffer Substances 0.000 description 1
- 235000010413 sodium alginate Nutrition 0.000 description 1
- 239000000661 sodium alginate Substances 0.000 description 1
- 229940005550 sodium alginate Drugs 0.000 description 1
- 235000019812 sodium carboxymethyl cellulose Nutrition 0.000 description 1
- 229920001027 sodium carboxymethylcellulose Polymers 0.000 description 1
- 159000000000 sodium salts Chemical class 0.000 description 1
- 230000003381 solubilizing effect Effects 0.000 description 1
- 238000000638 solvent extraction Methods 0.000 description 1
- 239000000600 sorbitol Substances 0.000 description 1
- 235000010356 sorbitol Nutrition 0.000 description 1
- 235000013599 spices Nutrition 0.000 description 1
- 238000001694 spray drying Methods 0.000 description 1
- 238000007619 statistical method Methods 0.000 description 1
- 229960005322 streptomycin Drugs 0.000 description 1
- 238000012916 structural analysis Methods 0.000 description 1
- 239000000829 suppository Substances 0.000 description 1
- 230000004083 survival effect Effects 0.000 description 1
- 239000000725 suspension Substances 0.000 description 1
- 230000002459 sustained effect Effects 0.000 description 1
- 238000013268 sustained release Methods 0.000 description 1
- 239000012730 sustained-release form Substances 0.000 description 1
- 230000008961 swelling Effects 0.000 description 1
- 208000024891 symptom Diseases 0.000 description 1
- 230000002195 synergetic effect Effects 0.000 description 1
- 239000000454 talc Substances 0.000 description 1
- 229910052623 talc Inorganic materials 0.000 description 1
- 239000012085 test solution Substances 0.000 description 1
- 235000010384 tocopherol Nutrition 0.000 description 1
- 229960001295 tocopherol Drugs 0.000 description 1
- 229930003799 tocopherol Natural products 0.000 description 1
- 239000011732 tocopherol Substances 0.000 description 1
- 238000012549 training Methods 0.000 description 1
- QORWJWZARLRLPR-UHFFFAOYSA-H tricalcium bis(phosphate) Chemical compound [Ca+2].[Ca+2].[Ca+2].[O-]P([O-])([O-])=O.[O-]P([O-])([O-])=O QORWJWZARLRLPR-UHFFFAOYSA-H 0.000 description 1
- 238000001291 vacuum drying Methods 0.000 description 1
- 230000035899 viability Effects 0.000 description 1
- 230000000007 visual effect Effects 0.000 description 1
- 239000011710 vitamin D Substances 0.000 description 1
- 235000019166 vitamin D Nutrition 0.000 description 1
- 150000003710 vitamin D derivatives Chemical class 0.000 description 1
- 229940046008 vitamin d Drugs 0.000 description 1
- 239000003643 water by type Substances 0.000 description 1
- 230000004584 weight gain Effects 0.000 description 1
- 235000019786 weight gain Nutrition 0.000 description 1
- CMFRFQODFZBKTI-UHFFFAOYSA-L zinc;4-benzamido-2,5-diethoxybenzenediazonium;tetrachloride Chemical compound [Cl-].[Cl-].Cl[Zn]Cl.CCOC1=CC([N+]#N)=C(OCC)C=C1NC(=O)C1=CC=CC=C1.CCOC1=CC([N+]#N)=C(OCC)C=C1NC(=O)C1=CC=CC=C1 CMFRFQODFZBKTI-UHFFFAOYSA-L 0.000 description 1
- GVJHHUAWPYXKBD-IEOSBIPESA-N α-tocopherol Chemical compound OC1=C(C)C(C)=C2O[C@@](CCC[C@H](C)CCC[C@H](C)CCCC(C)C)(C)CCC2=C1C GVJHHUAWPYXKBD-IEOSBIPESA-N 0.000 description 1
Images
Classifications
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K31/00—Medicinal preparations containing organic active ingredients
- A61K31/33—Heterocyclic compounds
- A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
- A61K31/357—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having two or more oxygen atoms in the same ring, e.g. crown ethers, guanadrel
- A61K31/36—Compounds containing methylenedioxyphenyl groups, e.g. sesamin
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23C—DAIRY PRODUCTS, e.g. MILK, BUTTER OR CHEESE; MILK OR CHEESE SUBSTITUTES; MAKING THEREOF
- A23C9/00—Milk preparations; Milk powder or milk powder preparations
- A23C9/12—Fermented milk preparations; Treatment using microorganisms or enzymes
- A23C9/13—Fermented milk preparations; Treatment using microorganisms or enzymes using additives
- A23C9/1307—Milk products or derivatives; Fruit or vegetable juices; Sugars, sugar alcohols, sweeteners; Oligosaccharides; Organic acids or salts thereof or acidifying agents; Flavours, dyes or pigments; Inert or aerosol gases; Carbonation methods
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F5/00—Coffee; Coffee substitutes; Preparations thereof
- A23F5/24—Extraction of coffee; Coffee extracts; Making instant coffee
- A23F5/243—Liquid, semi-liquid or non-dried semi-solid coffee extract preparations; Coffee gels; Liquid coffee in solid capsules
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23F—COFFEE; TEA; THEIR SUBSTITUTES; MANUFACTURE, PREPARATION, OR INFUSION THEREOF
- A23F5/00—Coffee; Coffee substitutes; Preparations thereof
- A23F5/24—Extraction of coffee; Coffee extracts; Making instant coffee
- A23F5/36—Further treatment of dried coffee extract; Preparations produced thereby, e.g. instant coffee
- A23F5/40—Further treatment of dried coffee extract; Preparations produced thereby, e.g. instant coffee using organic additives, e.g. milk, sugar
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/32—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G1/00—Cocoa; Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/30—Cocoa products, e.g. chocolate; Substitutes therefor
- A23G1/32—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds
- A23G1/48—Cocoa products, e.g. chocolate; Substitutes therefor characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, extracts
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23G—COCOA; COCOA PRODUCTS, e.g. CHOCOLATE; SUBSTITUTES FOR COCOA OR COCOA PRODUCTS; CONFECTIONERY; CHEWING GUM; ICE-CREAM; PREPARATION THEREOF
- A23G3/00—Sweetmeats; Confectionery; Marzipan; Coated or filled products
- A23G3/34—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof
- A23G3/36—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds
- A23G3/48—Sweetmeats, confectionery or marzipan; Processes for the preparation thereof characterised by the composition containing organic or inorganic compounds containing plants or parts thereof, e.g. fruits, seeds, extracts
-
- A23L1/3002—
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L2/00—Non-alcoholic beverages; Dry compositions or concentrates therefor; Their preparation
- A23L2/52—Adding ingredients
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L33/00—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof
- A23L33/10—Modifying nutritive qualities of foods; Dietetic products; Preparation or treatment thereof using additives
- A23L33/105—Plant extracts, their artificial duplicates or their derivatives
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K36/00—Medicinal preparations of undetermined constitution containing material from algae, lichens, fungi or plants, or derivatives thereof, e.g. traditional herbal medicines
- A61K36/18—Magnoliophyta (angiosperms)
- A61K36/185—Magnoliopsida (dicotyledons)
- A61K36/57—Magnoliaceae (Magnolia family)
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P19/00—Drugs for skeletal disorders
- A61P19/08—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease
- A61P19/10—Drugs for skeletal disorders for bone diseases, e.g. rachitism, Paget's disease for osteoporosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D493/00—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system
- C07D493/02—Heterocyclic compounds containing oxygen atoms as the only ring hetero atoms in the condensed system in which the condensed system contains two hetero rings
- C07D493/04—Ortho-condensed systems
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23L—FOODS, FOODSTUFFS, OR NON-ALCOHOLIC BEVERAGES, NOT COVERED BY SUBCLASSES A21D OR A23B-A23J; THEIR PREPARATION OR TREATMENT, e.g. COOKING, MODIFICATION OF NUTRITIVE QUALITIES, PHYSICAL TREATMENT; PRESERVATION OF FOODS OR FOODSTUFFS, IN GENERAL
- A23L7/00—Cereal-derived products; Malt products; Preparation or treatment thereof
- A23L7/10—Cereal-derived products
- A23L7/109—Types of pasta, e.g. macaroni or noodles
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2002/00—Food compositions, function of food ingredients or processes for food or foodstuffs
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
-
- A—HUMAN NECESSITIES
- A23—FOODS OR FOODSTUFFS; TREATMENT THEREOF, NOT COVERED BY OTHER CLASSES
- A23V—INDEXING SCHEME RELATING TO FOODS, FOODSTUFFS OR NON-ALCOHOLIC BEVERAGES AND LACTIC OR PROPIONIC ACID BACTERIA USED IN FOODSTUFFS OR FOOD PREPARATION
- A23V2200/00—Function of food ingredients
- A23V2200/30—Foods, ingredients or supplements having a functional effect on health
- A23V2200/306—Foods, ingredients or supplements having a functional effect on health having an effect on bone mass, e.g. osteoporosis prevention
Definitions
- the present invention relates to pharmaceutical composition for osteogenesis promotion comprising axial-equatorial aryl orientational furofuran type lignan, pharmaceutical preparation comprising thereof, functional food comprising thereof and health food comprising thereof.
- pharmaceutical composition for osteogenesis promotion comprising Fargesin and its derivatives, pharmaceutical preparation comprising thereof, functional food comprising thereof and health food comprising thereof.
- bone disease includes non-metabolic bone disease such as bone fracture and the like, and metabolic bone disease such as osteoporosis, bone Paget's disease, osteomalacia and the like.
- non-metabolic bone disease such as bone fracture and the like
- metabolic bone disease such as osteoporosis, bone Paget's disease, osteomalacia and the like.
- Several bone disease are caused by an inflammatory arthritis such as osteoarthritis, rheumatoid arthritis, and the like. Rheumatoid arthritis may cause periarticular site of osteoporosis.
- osteoporosis The metabolic bone disease, osteoporosis, is roughly classified into primary osteoporosis, which is not caused by other disease, and secondary osteoporosis, which is caused by other disease such as malignancy, rheumatoid, and others.
- primary osteoporosis accounts for 95% of entire of osteoporosis.
- osteoporosis has type I of which rate of crises is 6 times higher in women than men, and type II which generally develops in the patient over 60 years old.
- osteoclast cells which plays a role in the bone metabolism, is activated. As a result, bone resorption is enhanced, and bone density is reduced. This is caused by increase of cytokine levels derived from decreased estrogen secretion from ovary. Therefore, ⁇ -estradiol, a kind of estrogen, is uses as a prophylaxis and/or treatment agent for osteoporosis.
- the bone fracture as the non-metabolic bone disease is resulting from the large force loading onto the bone having normal strength at one stroke in a healthy person. In contrast, it is resulting from less force loading, which does not cause the bone fracture in the healthy person; onto the bones weaken by cancer, osteoporosis and the like. It is referred to as pathological fracture.
- the bone fracture is caused by repeated load onto the same place from exercise. It is referred to as “stress fracture”. It is said that the stress fracture is sometimes developed on metatarsal bone. As compared to men athletes, women athletes prone to breaking bones than men. As one of the reasons, there is mentioned that incidence rate of osteoporosis is higher in the women athletes than men athletes.
- An index of the metabolic bone disease abnormal blood calcium level is employed, because released amount of calcium into the blood is increasing after the development of osteoporosis.
- active vitamin D 3 as a derivative of vitamin D which plays an important role in calcium metabolism, calcitonin and derivative thereof, hormonal agent such as ⁇ estradiol and the like, and a variety of calcium compound and preparations are clinically used. It is known that among them, vitamin D 3 approaches the osteoclast cells and osteoblast cells, proto cells or precursor cells thereof to promote their proliferation.
- Fargesin is a lignan, and it is classified as the axial-equatorial aryl orientational furofuran type one based on its structure (patent document 2).
- Fargesin described on the reference is extracted from M. fargesii belonging to Magnoliaceae or Thurber's Magnolia ( Magnolia kobus DC. var. borealis Sarg.).
- Calcitonin and the derivative thereof, the hormone agent such as ⁇ -estradiol and the like are used for treating the bone disease.
- these agents sometimes cannot to be administrated to the patient, depending on absorption in vivo or metabolic problem. Also, there is the problem that it lacks predictability, because their receptor levels are highly individual.
- the treatment of the bone fracture is performed by surgical operation or reset in principle, except administration of an analgesic agent. Since osteoporosis causes the bone fracture, the treatment of osteoporosis and prevention are necessary to prevent the bone fracture.
- the first aspect of the present invention is a pharmaceutical composition for promoting osteogenesis comprising at least a substance selected from the group consisting of a axial-equatorial aryl orientation furofuran type lignin compound shown in the following chemical formula (I), a pharmacologically acceptable salt thereof, a pharmacologically acceptable hydrate thereof, and a pharmacologically acceptable glycoside thereof.
- R 1 and R 4 independently show one of functional group selected from the group consisting of a hydrogen atom, alkyl group having a carbon number 1 to 3, hydroxyl group, alkoxy group having the carbon number 1 to 3; R 2 and R 3 independently show one of functional group selected from the group consisting of alkyl group having a carbon number 1 to 3.
- the pharmaceutical composition for promoting osteogenesis preferably comprises at least a substance selected from the group consisting of a compound shown in the following chemical formula (II), a pharmacologically acceptable salt thereof, a pharmacologically acceptable hydrate thereof, and a pharmacologically acceptable glycoside thereof.
- the pharmaceutical composition is preferably applied for osteoporosis, hypercalcemia, hyper-parathyroid hormonemia, bone Paget's disease, arthritis, rheumatoid arthritis, metastasis of mammary cancer, osteomalacia, malignancy, and nutrition disorder, traumatic bone fracture, stress fracture or the like. Particularly, it is preferably applied to osteoporosis.
- the second aspect of the present invention is a pharmaceutical composition for promoting osteogenesis comprising an extract fraction obtained from one organ selected from the group consisting of a flower bud, leaf, cortex and xylem of Magnoliaceae plant, of which fraction containing the compound shown in the above-mentioned formula (II).
- the organ selected from the group consisting of the flower bud, leaf, cortex and xylem of Magnoliaceae plant is preferably obtained from the plant selected from the group consisting of Tamushiba ( Magnolia salicifolia Maximowicz), Kobushi ( Magnolia kobus De Candolle, Magnolia biondii Pampanini, Magnolia sprengeri Pampanini), Hakumokuren ( Magnolia heptapeta Dandy ( Magnolia denudata Desrousseaux) (Magnoli-aceae), and Kitakobushi ( Magnolia praecocissima var. borealis ).
- Tamushiba Magnolia salicifolia Maximowicz
- Kobushi Magnolia kobus De Candolle, Magnolia biondii Pampanini, Magnolia sprengeri Pampanini
- Hakumokuren Magnolia heptapeta Dandy ( Magnolia denudata Desrousseaux) (Magnoli-aceae)
- Kitakobushi Magnolia praecocis
- the third aspect of the present invention is the pharmaceutical preparation comprising the above-mentioned pharmaceutical composition as the active ingredient to be administrated at a predetermined dosage.
- the predetermined dose is preferably 10 to 350 mg/day in compound equivalent, more preferably 20 to 175 mg/day.
- the pharmaceutical preparation is preferably applied for osteoporosis, hypercalcemia, hyper-parathyroid hormonemia, bone Paget's disease, arthritis, rheumatoid arthritis, metastasis of mammary cancer, osteomalacia, malignancy, and nutrition disorder, traumatic bone fracture, stress fracture or the like. Particularly, it is preferably applied to osteoporosis.
- the fourth aspect of the present invention is the functional food comprising the composition of the first and/or the second aspect at the predetermined content.
- the fifth aspect of the present invention is the functional food comprising the composition of the first and/or the second aspect at the predetermined content.
- the food is more preferably a functional food or the health food for promoting the osteogenesis.
- the predetermined content is preferably 1 to 1,000 mg/kg.
- the amount intake of the functional food or the health food is preferably 10 to 350 mg/day expressed in terms of the amount of the above-mentioned compound, and more preferably 20 to 175 mg/day.
- the functional food or the health food may be preferably used to the bone disease such as osteoporosis, more preferably used to osteoporosis.
- the food may be cookies and biscuits, wheat and miscellaneous cereals for being supplemented to rice, noodles such as Japanese wheat noodle, soba noodle, and pasta, dairy product such as cheese, yogurt, jam, mayonnaise, processed soy product such as soybean paste, soy source, tea, coffee and cocoa, nonalcoholic beverage such as soft drinks and fruits juice, alcoholic beverage such as medicated liquor, snacks such as candy (drops), and chocolate, chewing gum, Japanese cracker, sweets made from azuki-bean such as azuki-bean jelly, to produce the functional food.
- noodles such as Japanese wheat noodle, soba noodle, and pasta
- dairy product such as cheese, yogurt, jam
- mayonnaise processed soy product
- nonalcoholic beverage such as soft drinks and fruits juice
- alcoholic beverage such as medicated liquor
- snacks such as candy (drops)
- chocolate chewing gum
- Japanese cracker sweets made from azuki-bean such as azuki-bean jelly
- the sixth aspect of the present invention is a treatment method for osteogenesis comprising the step for non-parenterally or parenterally administrating any one selected from the group consisting of the composition of the first aspect of the present invention, that of the second aspect of the present invention, and the pharmaceutical preparations to the patient necessary for promoting the cortical bone formation or the cancellous bone formation.
- composition or the pharmaceutical preparation is preferably taken orally; more preferably, it is combined with the exercise therapy, because fixing ration of calcium to the bone is improved.
- compositions, pharmaceutical preparation or foods, or the active ingredients or compositions used in the treatment method have sufficient advantageous effects for the bone density enhancement or the bone growth acceleration, and another advantageous effect to prevent and/or treat the bone diseases. Therefore, they have few side effects.
- FIG. 1 is a graph showing 1 H NMR specturm (400 MHz, CDCL 3 ) of fargesin.
- FIG. 2 is the graph showing 13 C NMR spectrum (400 MHz, CDCL 3 ) of fargesin.
- FIG. 3A shows measurement site in a femur.
- FIG. 3B shows a section of the measurement site in the femur.
- FIG. 4 is the graph showing a bone density (mg/cm 3 ) of the total bone, when a test substance is administrated to an ovariectomized mouse.
- FIG. 5 is the graph showing a bone density (mg/cm 3 ) of the cancellous bone, when a test substance is administrated to an ovariectomized mouse.
- FIG. 6 is the graph showing a bone density (mg/cm 3 ) of the cortical bone, when a test substance is administrated to an ovariectomized mouse.
- FIG. 7 is an image of transmitted light of the bone (left column) and a fluorescence staining image of the bone by using calcein (right column).
- FIG. 8A is the graph showing ALP activities and TRAP activities by using a relative ratio to a negative control (%), when the test substance is administrated to an ovariectomized mouse.
- FIG. 8B is a double stained image of ALP and TRAP, when the substance is administrated to co-cultured cells.
- FIG. 9A is the graph showing ALP activities and results of MTT assay by using the relative ratio the negative control (%), when the test substance is administrated to osteoblast-like cells.
- FIG. 9B is the graph showing ALP stained image, when the test substance is administrated to the osteoblast-like cells.
- FIG. 10A is the graph showing ALP activities by using the relative ratio the negative control (%), when the test substance is administrated to calcified osteoblast-like cells.
- FIG. 10B is the graph showing ALP stained image, when the test substance is administrated to the calcified osteoblast-like cells.
- FIG. 10C is the graph showing stained image of mineral deposition, when the test substance is administrated to the osteoblast-like cells.
- FIG. 11A is the graph showing the bone density in the mice of each group, a pseudo-operation group (Sham), or an ovariectomized group (OVX), after 3 month from the administration, when each substance was administrated to the mice.
- FIG. 11B is a figure showing the cancellous bone density of mice in the groups in shown in FIG. 11A .
- FIG. 12 is a figure showing polar coordination strength of mouse femur in the groups shown in FIG. 11A .
- FIG. 13 is a figure showing values TRACP5b of mouse sera of the groups shown in FIG. 11A .
- FIG. 14A is a graph showing the total bone density of the mice of the sRANKL administration or the test sample administration group after 13 days from administration.
- FIG. 14B is the graph showing the mouse cancellous bone density of the group shown in FIG. 14A .
- FIG. 15 is the graph showing the polar coordination strength of mouse femur in the groups shown in FIG. 14A .
- the first aspect of the present invention is a pharmaceutical composition for promoting osteogenesis comprising at least a compound shown in the following formula (I), the axial-equatorial aryl orientational furofuran type lignin compound and their derivatives.
- R 1 and R 4 independently show one of functional group selected from the group consisting of a hydrogen atom, alkyl group having a carbon number 1 to 3, hydroxyl group, alkoxy group having the carbon number 1 to 3;
- R 2 and R 3 independently show one of functional group selected from the group consisting of alkyl group having a carbon number 1 to 3.
- the compound shown in the formula (I) is preferably that sown in the formula (II), because it has high promoting activities of bone density, growth of bones, formation of the cortical bone or cancellous bone.
- the derivative includes the physiologically acceptable salt thereof, hydrate thereof, and a glycoside thereof, and a mixture thereof.
- physiologically acceptable salt thereof there are mentioned such as sodium salt, potassium salt, chloride salt and the like.
- physiologically acceptable hydrate there are mentioned such as monohydrate, dehydrate and the like.
- the compound shown in the formulae (I) to (III) and analogs thereof, salt thereof, hydrate thereof, glycoside thereof, and mixture thereof may be prepared by using the conventional method or according to that to obtain. Commercially available ones may be purchased.
- composition of the present composition may be produced as described below.
- an organ selected from the group consisting of the flower bud, leaves, bark, and xylem is collected to dry to obtain dried ones.
- Boushunka dried flower bud of Magnolia biondii is prepared.
- the dried flower bud may be prepared to collect them from such plants by using air drying.
- the commercially available one sold as Shinni as the crude drug may be purchased to use. Instead of the flower but, the leaves, the bark or the xylem may be used.
- the dried one is weighted at a predetermined weight; then about 1.7 to 7 times volume of the weight of methanol is added to perform the extraction at the predetermined temperature. Solid content is separated by filtration from the extract. Then, methanol is removed and the weight of a residue is weighed. Two to five volumes of the residue weight of the mixture of water/ethyl acetate is added to the residue to perform a partitioned extraction at the predetermined temperature.
- the solid content is separated by using a device such as Buchner funnel and the like. Subsequently, solvent is removed by using a rotary evaporator, flush evaporator and the like.
- the amount of the residue is weighed, and then, for example, about 2 to 5 time volume of the mixture of water/organic solvent is added to be transferred into a separatory funnel. After that, it is subjected to the partitioned extraction at room temperature.
- a liquid-liquid extraction equipment, counter-current extraction equipment, and the like may be chosen, depending on the volume of the dried flower bud or other dried one.
- water/ethyl acetate, water/acetone, water/butanol, and the like may be used for the partitioned extraction.
- water/ethyl acetate is preferably used, because it is easier to remove the solvent from the organic phase.
- the organic phase is separated from the aqueous phase. Then, the organic solvent of the organic phase obtained is removed by using the evaporator and the like to obtain the first concentrated solution.
- the aqueous phase is separated from the organic phase. Then, the same organic phase to be separated is added to the organic phase at the same volume, and then separated. The procedure is repeated. By this, the intended compounds are extracted more, and the intended compound is obtained at high efficiency.
- the first concentrated solution is performed to the second partitioned extraction by using the different solvent system.
- the mixture of the organic solvent is added to the first concentrated solution at 2 to 5 times volume of the first concentrated solution to perform the second partitioned extraction at the predetermined temperature.
- the solvent system such as n-hexane/water, n-hexane/methanol and the like is preferably employed.
- n-hexane/methanol aqueous methanol including about 10% of water is preferably employed.
- the repeated extraction by using n-hexane has an advantageous effect that it accelerates elimination of the aliphatic components, and makes purification hereinafter easier. After that, obtained MeOH phase is separated similarly that as mentioned above to concentrate to obtain the second concentrated solution.
- composition for prophylaxis and/or treatment of the bone disease may be produced from the according to the conventional method by removing MeOH to obtain crystalline.
- an open column of diameter from 5 to 20 cm ⁇ length 12.5 or 75 cm is prepared, and 200 to 800 g of silica gel is packed into it.
- the first solvent is poured into the column to swell the silica gel.
- the second concentrated solution is applied on the gel to be fractionated by using a step gradient method to obtain the first fractions.
- the volume of the fractions may be properly decided, it is preferable to set the fraction as 0.75 to 1.5 L, because of the operability efficiency.
- 100% MeOH is employed for elution of the components adsorbed on the silica gel.
- the content of the intended component may be confirmed by using a thin layer chromatography.
- the mixture ratio of the elution solvent to have the highest yield is changed depending on the volume of dried flower bud, quality, solvent volume used for the subsequent extraction operation, the extraction temperature. Therefore, it is preferable to confirm the yield of each fraction by using the thin layer chromatography.
- the crystalline may be sometimes precipitated in the fraction including high content of the intended compound.
- the precipitates are separated by the filtration to be recrystallized according to the conventional method to obtain the crystalline with high purity.
- fractions are processed as the same as the above-mentioned concentrated solution to obtain the pharmaceutical preparation for osteogenesis promotion of the present invention.
- the first fractions of them are concentrated as the same as those described above, they are purified the following procedure by using the preparative chromatography.
- the concentrated solution of the first fractions is applied onto a reverse phase column chromatography by using the octadecyl silica column (C 18 -ODS), the inner diameter 2 cm ⁇ the length 20 cm; then fractionated by using the preparative chromatography.
- the elution solvent for example, water/methanol, of which mixture ratio are changed by 20%, may be employed.
- the purification may be performed by using the step gradient method.
- the fractions are collected to the same ratio, and they are concentrated, checking the intended compound content.
- the intended compounds are included in the 80% methanol fraction to give the precipitate as the crystalline by the concentration, the crystalline may be obtained by filtration of the concentrated fraction with, for example, No. 2 grade filter paper.
- the obtained crystalline is dissolved in the predetermined solvent, and they are subjected to mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR). Then, the obtained spectrum data are compared to the reference data to identify the structure of the obtained compound.
- MS mass spectrometry
- NMR nuclear magnetic resonance spectroscopy
- the pharmaceutical preparation for osteogenesis promotion By using thus obtained compound or the composition (partially purified fractions), the pharmaceutical preparation for osteogenesis promotion, the functional food and the health food may be produced.
- the optimal dose of the compounds or the compositions for the human administration is generally about 50 times of that for the mouse.
- when it is administrated to the body weight 20 g of the mouse as the dosage of 20 mg/kg body weight/day, or to the body weight 35 g of the mouse at the dosage form of 100 mg/kg body weight/day are respectively converted to the dosage of 20 mg/day or 175 mg/day in human.
- the second aspect of the present invention is the pharmaceutical preparation for osteogenesis promotion comprising the above-mentioned compound as the active ingredient.
- pharmaceutical preparations there are mentioned non-parenteral dosage from such as injections, suppositories, aerosols, percutaneous form and so forth, parenteral preparations such as tablets, powders, capsules, pills, trochiscus, solutions and so forth.
- the above-mentioned tablet includes sugar coated tablets, coat tablets, and buccal tablets; the capsule includes both of hard capsules and soft capsules.
- the granules contain coated granules.
- the above-mentioned solution contains suspensions, emulsions, syrups, elixirs, and so forth, and the syrup includes also dry syrups.
- compositions include the liquid formulation of the above-mentioned compositions, or the gel formulation preparation which is an impregnated gel the liquid form and the like. Note that the above-mentioned formulations include both of the sustained and non-sustained release formulation.
- preparations may be formulated according to the known pharmaceutical method by using pharmacologically acceptable carrier, excipient, disintegrator, lubricant, colorant, and so forth, for formulating the preparation, described on Japanese Pharmacopoeia.
- carriers or excipients for example, there are mentioned such as lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, powdered glycyrrhiza extract, powdered gentian, and so forth.
- disintegrator for example, there are mentioned such as starch, agar, powdered gelatin, sodium carboxymethylcellulose, calcium carboxymethylcellulose, crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate and so forth; as the lubricant, for example, there are mentioned such as magnesium stearate, talc, hydrogenated vegetable oil, macrogol and so forth.
- the colorant which is acceptable to be added to the pharmaceutical preparation, can be used with no limitation. Except these additives, a corrigent and so forth cam be used depending on the necessity.
- the tablet or the granule When formulating the tablet or the granule, if necessary, they may be coated by using sucrose, gelatin, hydroxypropylcellulose, purified shellac, gelatin, glycerin, sorbitol, ethylcellulose, hydroxy-propylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, acetate cellulose phthalate, hydroxypropylmethylcellulose phthalate, methylmethacrylate, methacrylate polymer, and so forth to have single coating or plural coatings.
- the capsule can be prepared by encapsulating the granule or powdered preparation into the capsule made of ethylcellulose, gelatin, and so forth.
- the physiologically acceptable salt thereof, or the hydrate thereof a PH regulator, a buffering agent, a stabilizer, a solubilizing agent, and so forth may be added as needed.
- the dosage is depending on conditions such as thickness of the symptom, age, weight, and health status and so forth.
- the preparation is administrated for an adult in the parenteral or non-parenteral route, at the dosage of 1 mg/kg to 2,000 mg/kg, preferably 1 mg/kg to 1,000 mg/kg once a day or more.
- it is preferably administrated at the dosage of 10 to 350 mg/day per an adult, and more preferably 20 to 175 mg/day per the adult. Number of administration and amounts a day may be adjusted depending on the conditions described above optionally.
- the extract obtained in the production process may be condensed, and dried by using the method such as lyophilization, spray-drying, vacuum-drying and so forth; and then dried extract is pulverized into fine powder.
- Corn starch, dextrin, cyclodextrin, oyster shell powder may be added as needed
- the above-mentioned binder is optionally added to the powder obtained as described above and compressed to from the tablet.
- the coating agent such as sucrose, gelatin and so forth to formulate the sugar-coated tablet, or coated by other coating agent to formulate enteric coated tablet.
- the powder obtained as describe above may be granulated by using the conventional method to formulate the granule.
- the powder or granule as mentioned above is encapsulated into capsules in a proper amount to formulate the capsule.
- compositions are added to, for example, breads, cookies, biscuits, wheat to be supplemented to rice and cereals, noodles such as Japanese wheat noodles, buckwheat noodles, pasta and others, dairy food such as cheese, yogurt and others, jam, mayonnaise, soy bean product such as soy bean paste, soy source and others, nonalcoholic beverage such as tea, coffee and cocoa, soft drinks such as and fruits juice, alcoholic beverage such as medicated liquor, snacks such as candy (drops), and chocolate, chewing gum, Japanese cracker, azuki-bean jelly and so forth, to produce the functional food or healthy food having preventing or treatment effect of periodontal disease.
- compositions is added to yogurt, soy source, drinks and the like, solubilizing auxiliaries or the stabilizers may be employed not so as to form crystalline of the composition of the present invention to precipitated.
- composition of the present invention may be employed solely, or as a combination of 2 or more to formulate the powders, the granules, the tablets or the capsules to produce the healthy food.
- the food taken by the bone disease patients for the predetermined period in predetermined number and amounts promotes the cancellous bone formation of them to effectively prevent the appearance of the bone disease such as osteoporosis and the like.
- mice of 4 weeks old (Japan SLC, Inc.) was operated with ovariectomy to use as a type I osteoporosis model animal. Also, in order to delete the affection by invasion, a group which is operated without ovariectomy (Sham operation) is set (Sham group). These were the aged model animals.
- the crude extract was poured into 5 L volume of the separatory funnel, and 2 L of water/ethyl acetate (1/1) was added.
- the funnel was shaken to perform the partitioned extraction.
- the obtained ethyl acetate phase was concentrated by using the evaporator to be poured into another 5 L of the separatory funnel.
- Two L of hydrous methanol (10% of water content ratio)/hexane was added into the funnel to perform the partitioned extract again.
- Fargesin was dissolved in 30 mL of a solution (it is referred to as “TD solution”, hereinbelow) containing 4% dimethyl sulfoxide (it is referred to as “DMSO”, hereinbelow) and 4% TWEEN 80 (both were purchased from Wako Pure Chemical Industries, Ltd.) so as that a dosage amount of fargesin becomes 20 mg/kg body wt/day, or 10 mg/kg body wt/day.
- TD solution a solution
- DMSO dimethyl sulfoxide
- TWEEN 80 both were purchased from Wako Pure Chemical Industries, Ltd.
- ⁇ -estradiol 4% dimethyl sulfoxide (refer to DMSO thereafter) was dissolved in 30 mL of an aqueous solution containing 2% DMSO to prepare ⁇ -estradiol solution so as that the dose amount of it becomes 100 ⁇ g/kg body wt/day.
- mice were adapted to the circumstances for 5 days, and then they were divided to the Sham group, the negative control, the positive control group, and the test substance administration group (6 mice per group).
- the ovariectomized mice were used for the negative control group, the positive control group, and the test substance administration group.
- mice with Sham operation group and the negative control group they were collectively referred to as “NC group”) p.o. for 3 months.
- ⁇ -estradiol solution was daily administrated to the ovariectomized mice at the dose of 100 ⁇ g/kg body wt/day (it is referred to as “B100 group” thereafter) i.p.
- Fargesin solution was daily administrated to the ovariectomized mice in the test group at the dosage of 20 mg/kg body wt/day (it is referred to as “F20 group”, hereinbelow) or 100 mg/kg body wt/day (it is referred to as “F100 group”, hereinbelow) for 3 month p.o.
- the femur In order to study effects of fargesin on the bone, the femur, the largest long bone, was used.
- mice in each group were killed by the cervical dislocation under diethyl ether anesthesia. Their right femurs were excised with muscles, and the muscles were removed from the femur after the excision. Lengths of the excised bones from the mice in the groups were measured, and they were dipped in 70% ethanol to be fixed.
- the bone was shown schematically.
- the long bone (tubular bone) was composed of two thick and rounded ends (epiphysis), and thin and long part between them (a shaft).
- the bone extends to longitudinal direction, depending on the growth of a plate-shape epiphyseal cartilage (a growth plate) between the epiphysis and the shaft.
- the region of 1 mm proximal side from the A position for a measurement was set to that from 1 mm proximal region (epiphysis) from the distal growth plate as decided as the measurement region.
- the bone density was measured by using the peripheral quantitative computed tomography (pQCT, XCT Research SA+, Stratec Medizintechnik GmbH, Merk Ltd) ( FIG. 3B ). The measurement was performed under the conditions: a diameter of 90 mm, a voxel size of 0.12 mm, a CT speed of 10 mm/sec, and a block number of 1. Bone mineral content (mg/mm), bone density (mg/cm 3 ), and cross section of the bone (mm 2 ) of the entire of tomographic images (whole bone area) were calculated.
- pQCT peripheral quantitative computed tomography
- bone mass means the sum of the bone mineral density and amount of protein substrate.
- the value of the bone mineral amount divided by the section of the bone is bone mineral amount per unit volume (the bone density).
- the area of the cancellous bone was extracted (peel mode 20), and then the bone mineral amount (mg/mm), the section of the bone (mm 2 ), and the bone density (mg/cm 3 ) were calculated. Further to the cortical bone, the bone mineral amount (mg/mm), the bone density (mg/cm 3 ), the section of the bone (mm 2 ), a bone thickness (mm), the periosteum perimeter of the cortical bone (mm), and the endosteum perimeter of the cortical bone (mm) were calculated.
- SSI was calculated.
- SSI is composed of Polar SSI (torsion strength in polar coordination), X-axis SSI (strength in X axis, three-point bending strength), and Y-axis SSI (Y axis strength, three-point lateral bending strength), and it is expressed by the following general equation.
- Z is the coefficient of the cross section (mm 3 )
- CBD is the bone density of the cortical bone (mg/cm 3 )
- ND is the physiological bone density (1200 mg/cm 3 ).
- the coefficient Z is shown as the following equation.
- the measurement sites were as mentioned above.
- a direction from the proximal epiphysis to the distal epiphysis is defined as the polar direction
- X axis direction is defined as a horizontal direction away from the body axis
- Y axis direction is defined as vertically downward direction ( FIGS. 3A and B).
- the polar coordinate torsion strength, X axis strength, and Y axis strength were respectively calculated.
- Makejob Stratec Medizintechnik GmbH., Germany
- mice in each group were 19.8 g to 21.2 g at 4-week-old, 29.2 to 31.5 g at 8-week-old. At 12 week-old, the mice became almost mature and their weights reached 32.1 to 36.4 g. Average weight of B100 group mice was lower than that of NC group mice during mouse growth. However, the average weights of F20 group mice and F100 group mice were equal to that of NC group mice during the mouse growth.
- the bones were classified into the whole bone, the cancellous bone, and the cortical bone (a compact bone), the change of the bone mineral mass, the cross-section area, and the bone density were studied. Results are shown in Tables 1 and 2, and FIGS. 4 and 5 .
- Tables the number written in lower side in each column shows relative value, when the data of Sham group is 100.
- the data was statistically processed by using Dennett's two-sided t test to decide whether there is significant difference or not. Each number in the table was shown in average ⁇ standard error. From Tables 3 and after that were the same as Tables 1 and 2.
- the bone mineral mass 0.6333 ⁇ 0.077 mg/mm, the cross section areas of the bone 1.787 ⁇ 0.091 mm 2 , the bone density 350.750 ⁇ 24.892 mg/mm 3 were as follows: the bone mineral mass was 0.354 ⁇ 0.091 mm 2 , the cross sectional area of the bone was 1.606 ⁇ 0.058 mm 2 , and the bone density was 216.080 ⁇ 24.892 mg/mm 3 , and the trends were the same as those in the whole bone B100 group whose bone mineral mass is 0.725 ⁇ 0.110 mg/mm and the bone density was 399.050 ⁇ 53.851 mg/cm 3 , and they showed increased trend, but they did not show any significant difference between them.
- the bone mineral mass was 0.855 ⁇ 0.067 mg/mm, and the bone density was 431.283 ⁇ 30.506 mg/cm 3 .
- the bone mineral mass was 0.920 ⁇ 0.102 mg/mm and the bone density was 460.500 ⁇ 36.222 mg/cm 3 .
- the bone mineral mass and the cross section area of both groups were significantly increased (Table 2, FIGS. 4 and 5 ). Therefore, it was demonstrated that fargesin maintained the bone mineral mass of the cancellous bone, which would be decreased by the ovariectomization, and increase the bone density.
- the bone mineral mass was 2.192 ⁇ 0.180 mg/mm
- the cross section area was 2.477 ⁇ 0.182 mm 2
- the bone density was 822.083 ⁇ 12.665 mg/cm 3
- the bone thickness was 0.361 ⁇ 0.023 mg/mm.
- the bone mineral mass was 1.150 ⁇ 0.246 mg/mm
- the cross section area of the bone was 1.390 ⁇ 0.277 mm 2
- the bone density was 826.540 ⁇ 11.518 mg/cm 3
- the bone thickness was 0.206 ⁇ 0.040 mg/mm in the NC group.
- the NC group since all of the values were significantly decreased. Particularly, the bone mineral mass, the cross section of the bone, and the bone thickness were severely decreased, it was demonstrated that the cortical bone became thinner and brittler.
- the bone mineral mass was 2.163 ⁇ 0.230 mg/mm
- the cross section area of the bone was 2.4825 ⁇ 0.230 mm 2
- the bone density was 867.475 ⁇ 13.724 mg/cm 3
- the bone thickness was 0.365 ⁇ 0.038 mg/mm in the F100 group.
- the bone mineral mass was 2.087 ⁇ 0.160 mg/mm
- the cross section area of the bone was 2.427 ⁇ 0.185 mm 2
- the bone density was 859.483 ⁇ 2.479 mg/cm 3
- the bone thickness was 0.329 ⁇ 0.026 mg/mm in the F20 group.
- the bone mineral mass was 2.240 ⁇ 0.320 mg/mm
- the cross section area of the bone was 2.640 ⁇ 0.361 mm 2
- the bone density was 843.55 ⁇ 8.714 mg/cm 3
- the bone thickness was 0.370 ⁇ 0.051 mg/mm.
- the bone mineral mass, the bone density, and the cross section area of the bone were significantly increased.
- fargesin had effect for preventing the brittle cortical bone at the same level as that of ⁇ -estradiol. Further, it was demonstrated that there were a tendency for fargesin to have was highly effects than those of ⁇ -estradiol for boundary lengths of the bone adventia and bone endosteum (Table 2).
- Polar axis strength (polar coordinates strength) was 1.599 ⁇ 0.143 mm 3 in the Sham group, and 1.145 ⁇ 0.129 mm 3 in the NC group. This showed that the bone strength index decreased in the NC group. On the other hand, it was 1.376 ⁇ 0.088 mm 3 in the B100 group. There was the tendency for the NC group to decrease the strength; however, there was no significant difference between those groups.
- fargesin largely increases the bone strength index than ⁇ -estradiol does. This shows that fargesin effectively prevents the brittle bone caused by the ovariectomy.
- MEM Eagle's minimum essential mediums
- GEBCO penicillin/streptomycin
- fetal bovine serum Sigma-Aldrich Co. LLC.
- 50 ⁇ g/ml of ascorbic acid Wako Pure Chemical Industries, Ltd.
- 1 mM of ⁇ -glycerophosphoric acid Sigma-Aldrich Co. LLC.
- 1 ⁇ g/ml of calcein Sigma-Aldrich Co. LLC.
- ⁇ M of fargesin As a test substance, 0.3 ⁇ M of fargesin, or as a control, 0.3% of DMSO (final cone., Wako Pure Chemical Industries, Ltd.) was added to the organ culture medium. Fargesin was used the same solution prepared in Example 1 containing 0.3% DMSO as the final concentration.
- mouse fetuses were took from pregnant female ICR mice (Japan SLC, Inc.) by Caesarean operation (E15.5) from the 15 th days of a pregnant ICR female mouse by Caesarean section. Left and right metatarsals of the fetus were excised to be placed into the organ culture medium. The organ culture was performed under the conditions of 5% CO 2 at 37 degree centigrade for 7 days to observe the effect on long axis direction.
- the long axis of the bone grows depending on that of epiphyseal cartilage existing between an epiphysis and a shaft.
- calcium salt resulted in deposition of the substances around the cartilage cells.
- calcification was occurred from the occification center (ossification).
- the calcified region was fluorescently-stained by using calcein, and then observed under the microscope. The observed image was shown in FIG. 7 .
- mice Four-week-old ddY male mice (Japan SLC, Inc.) were killed by the cervical dislocation, and then, the long bones of left and right lower extremities were excised with muscles. All of the muscles were removed from the bones, and the femurs and shinbones were obtained. The both ends of the obtained femurs and shinbones were scraped a little by a little.
- the cells in the bone morrow were pushed out into the following medium by using a 2.5 ml of syringe with a needle (22G ⁇ 11 ⁇ 4; Thermo corporation). After that, contaminants were eliminated through a filter (70 ⁇ M Nylon Cell Strainer; Japan Becton, Dickinson and Company) to obtain more than 2 ⁇ 10 8 cells of BMCs.
- Osteoblast-like cells UAMS-32 cells, were purchased from the Institution of Physical and Chemical Research (Japan).
- BMCs were plated at the concentration of 2 ⁇ 10 6 cells/well, and UAMS-32 cells were plated at the concentration of 1 ⁇ 10 5 cells/well in each well of the 96 well plates, respectively. Then, the cells were co-cultured in the co-culture medium under the conditions of 5% CO 2 and 37 degree centigrade for 5 days. The medium were changed on the 3 rd days from the culture start. After cells were fixed as described in below, alkaline phosphatase (ALP) activity was measured as the index of osteoblast differentiation; tartrate-resistant acid phosphatase activity was measured as the index of osteoclast differentiation.
- ALP alkaline phosphatase
- the cells were cultured under the above-mentioned conditions, 10% formalin aqueous solution was added into each well still standing for 10 minutes, and then ethanol was added still standing for further 1 minute to fix the cells.
- 10 mM sodium tartrate/50 mM citric acid buffer solution containing 1.36 mg of p-nitrophenyl sodium phosphate (Sigma-Aldrich Co. LLC.) (pH 4.6) was prepared as a substrate solution for TRAP.
- the substrate solution for TRAP was added to the well at the volume of 100 ⁇ l/well, and reacted for 15 to 20 minutes at a room temperature.
- the reaction solution was transferred into another 96 well plate, to which 100 ⁇ l/well of 0.1N NaOH was previously added to stop the reaction, and they were measured at absorbance of 405 nm.
- TRAP staining solution 50 mM sodium tartrate/0.1 M of sodium acetate buffer (pH 5.0) including 0.1 mg/ml naphthol AS-MX phosphate (sigma N-4875) and 0.6 mg/ml Fast red violet LB salt (both from Sigma-Aldrich Japan Co. LLC.) was prepared as TRAP staining solution.
- the TRAP staining solution was added to the wells to stain the cells in negative control group till they took on red color under room temperature. After that, the cells were washed with distilled water. The cells stained red and having not less than two nuclei was decided as multinuclear osteoclast, of which number was counted by using the microscope.
- ALP substance solution was added with the volume of 100 ⁇ l/well, and then the reaction was performed at room temperature for 15 to 20 minutes. Next, the reaction solution was transferred into another 96 well plate being added 100 ⁇ l/well of 0.1N NaOH to stop the reaction. Then, the plate was measured the absorbance at 405 nm, and set the value as the index of ALP activity.
- the reaction was performed at room temperature, until the cells of the negative control group were colored blue-violet. Then, the cells were washed. The strength of the staining was determined by visual observation to determine the ALP activity.
- fargesin activated the osteoblast; on the other hand, it inhibited the promotion of the differentiation from the osteoclast precursor cells to the osteoclast by the osteoblast.
- MC3T3-E1 cells ((IAA) the Institution of Physical and Chemical Research), osteoblast-like cell line derived from the mouse fetus cranial bone cells, were inoculated into the 96 well plate at 4,000 cells/well.
- the basal medium prepared in the Example 3 supplemented with fargesin at 2 to 80 ⁇ M was used as a medium.
- Pre-culture was performed by using the basal medium only under the conditions of 5% CO 2 and 37 degree centigrade for 3 days. After that, the conditioned medium was changed to fresh medium with fargesin; the culture was performed under the conditions of 5% CO 2 and 37 degree centigrade for 3 days.
- the conditioned medium was changed to the fresh medium; the culture was further performed under the conditions of 5% CO 2 and 37 degree centigrade for 3 days. After the termination of the culture, the following MTT test, measurement of ALP activities, and the staining were performed.
- MTT test was measured by using MTT test.
- MTT reagent was prepared by dissolving 50 mg of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide in 10 ml of PBS ( ⁇ ). After the termination of the culture, the medium containing fargesin was partially removed from each well to adjust the solution volume to 100 ⁇ L. Ten ⁇ l of the MTT reagent was then added to each well to be reacted to give blue-violet color in the CO 2 incubator.
- Measurement of the ALP activity, statistical processing of the data, and the observation of differentiated cells were performed as the same way as those in Example 3, except the staining of the osteoclast cells were omitted.
- ALP activity and the cell viability were shown in Table 8 as relative values when the negative control was 100.
- MC3T3-E1 cells as the same as those used in Example 4 were used.
- 50 ⁇ g/ml of L-ascorbic acid and 10 mM ⁇ -Glycerophosphate (both from Sigma-Aldrich Japan Co. LLC.) were added to the basal medium employed in the Example 3 was used.
- Fargesin prepared in the Example 1 was added to the medium the concentration at 2 to 80 ⁇ M.
- MC3T3-E1 was inoculated in each well of the 96 well plates at 4,000 cells/well, and then the plate was pre-cultured by using only basal mediums under the condition of 5% CO 2 and 37 degree centigrade for 2 days. After that, the medium was exchanged to the medium containing 50 ⁇ g/ml of L-ascorbic acid, 10 mM ⁇ -Glycerophosphate, and fargesin, and it was incubated under the condition of 5% CO 2 and 37 degree centigrade for 5 days. After the termination of the incubation, the measurement of ALP activity and the differentiated cells were observed as the same as those in Example 3.
- ALP activity was shown in Table 6 as relative values, when the negative control group was 100.
- mice Four-week-old female Slc; ddy mice (Japan SLC, Inc.) were purchased, and they were divided into 8 groups without acclimate keeping (6 mice per group). They were anesthetized by using 50 mg/kg of pentobarbital to be ovariectomized (it is referred to as “OVX” herein below) or to have Sham operation (it is referred to as “Sham” herein below) under the anesthesia which is given by intraperitoneal administration of 50 mg/kg of Somnopentyl (pentobarbital sodium).
- each group was maintained under the condition of 12 hour light/dark cycle, 23 ⁇ 3 degree centigrade as the room temperature, and humidity 55 ⁇ 5% for 2 months as the bone resorption induction period.
- the animal bedding was exchanged twice a week and all fresh bedding was used at all times.
- CRF-1 Oriental Yeast Co., Ltd.
- test substance After two month period from the test start, the bone resorption induction period, the test substance were given to each group at the amount shown in the following Table 7 for 3 month.
- Human PTH (1-34) (herein below, it is sometimes referred to as “hPTH (1-34)”) were dissolved in distilled water at the concentration of 80 ⁇ g/kg/day, and it was administrated subcutaneously.
- mice belonging to all of the groups were weighed. They were anesthetized by using diethylether, and were collected their blood from their hearts, and then they were died. Then, organs such as the uterus, the white adipose tissue (WAT), the brown adipose tissue (BAT), the liver, the spleen, and both left and right lower extremities were removed. The left and right lower extremities were separated into the femurs and the tibias, and they were stored in 70% EtOH under the room temperature.
- WAT white adipose tissue
- BAT brown adipose tissue
- the muscles were removed from the obtained femur (right), and then its bone density and the like were measured by using pQCT employed in Example 1 under the following conditions.
- CORTMODE used in CORTBD 1* * Voxels not larger than the standard value for distinguishing the cortical bone from the cancellous bone (threshold: threshold) were omitted. Each tissue weight was also measured.
- the weights of the body, the uterus, BAT, WAT, the liver, and the spleens of each group were weighed. There were no biases depending on the groups. Also, since the weight gains of the uterus, the inhibition of atrophy, in both of PTH administration group and fargesin administration group were not shown, it was considered that these compounds did not have estrogen-like functions.
- the amount of feed intake (weight) of Sham group was slightly much than that of the OVX group. However, there was not much difference between them.
- both of the whole bone densities and the cancellous bone densities were higher than those in control OVX group.
- Significant recovery of the bone mass was observed.
- the cancellous bone density was largely increased.
- the cancellous bone density was dose-dependently increased in the fargesin administration group. As compared to the control OVX group, it was significantly increased in fargesin administration group.
- the cancellous bone density of 90% MeOH fraction administration group was higher than that of Far 2 group, and it indicated the possibility of synergic effects with chemical compounds included in the fraction other than fargesin.
- the cortical bone mineral content and the cortical bone density were significantly decreased in the Control OVX group compared to that of the Sham OVX group as the same as those of the whole bone and the cancellous bone.
- the cortical bone content was remarkably decreased. Differently from the cases of the whole bone and the cancellous bone, the bone content was not restored by the administration of PTH. It was also not significantly restored by fargesin administration or 90% MeOH fraction administration (see Table 9).
- Osteogenesis mainly occurs in the bone adventitia side, and the decrease of the cortical bone mass was appears as extension of Haversian canal (porous formation in the cortical bone).
- boundary length of the cortical bone adventitia reflects the osteogenesis (Biomedical Engineering vol. 44, No. 4: 517-521, 2006, ibid. vol. 44, No. 4: 490-502, 2006), and the increase of the length of the cortical bone endosteum reflects the bone resorption. Therefore, it was shown that the bone resorption was increased and the osteogenesis was decreased in the OVX operation group. In contrast, there was the trend that both of the osteogenesis and bone resorption were increased together in PTH administration. It was considered that the bone mass was increased because of dominant osteogenesis. In fargesin administration group, there was the trend that both of the osteogenesis and bone resorption were dose-dependently increased.
- Bone strength in the Control OVX group was significantly decreased as compared to that in the Sham OVX group.
- the bone strengths were significantly increased in both of the PTH administration group and the fargesin administration group (2 mg and 40 mg administration groups) as compared to that of the Control OVX group.
- the bone strength in the 90% MeOH fraction administration group was higher than that in the Control OVX group; however, there was no significant difference between them (see FIG. 12 ).
- TRACP tartrate-resistant acid phosphatase
- TRACP 5b value in 90% MeOH fraction administration is higher than that in Far 2 group, indicating interrelated perimeters of the cortical bone lining membranes.
- Serum TRACP5b in Fargesin administration group is decreased, showing concentration dependent and is significantly decreased in 40 mg administration group. Thus, it shows that Fargesin restrains the osteoclast from activating and increasing the number, which is the same result as RAW264.7 ( FIG. 13 ).
- the level of TRACP 5b did not show major alteration in hPTH administration group; however, it showed the correlation with the length of the cortical bone endosteum.
- TRACP 5b level in the 90% MeOH fraction administration group was higher than that of Far2 group, and it showed the correlation with the length of the cortical bone endosteum.
- fargesin administration group the level of TRACP5b in the sera was dose-dependently decreased, and it was significantly decreased in the 40 mg administration group. As a result, it was demonstrated that fargesin inhibited the activity of the osteoclast cells and the cell number, similarly to the case in which RAW264.7 was employed (see FIG. 13 ).
- C57BL/6NCrlCrlj mice (6-week-age, female) were purchased from Oriental Yeast Co., Ltd. and kept to be acclimatized under the conditions of 12 hour light/dark cycle, 23 ⁇ 3 degree centigrade, and humidity 55 ⁇ 5% for 7 days. Two mice were in a cage, and the animal bedding was changed twice a week, and all fresh animal bedding was used at all times. As the feed, CRF-1 (Oriental Yeast Co., Ltd.), and deionized water as the drinking water were freely taken.
- mice were randomly divided into 4 groups (8 mice per group), sRANKL administration group and fargesin administration groups.
- Fargesin administration groups had 0.2 mg/kg/day administration group (it is referred to as “Far 0.2”, hereinbelow), 2 mg/kg/day administration group (it is referred to as “Far 2” herein below), and 20 mg/kg/day administration group (it is referred to as “Far 20” herein below).
- sRANKL (1 mg/kg/day, i.p) was administered to all mice on the first day and the second day from test start to cause osteopenia experimentally.
- distilled water or the test compounds shown in the following Table 10 were daily administered to the mice in each group.
- the femurs were removed in the same way as employed in Example 6 to be excised to evaluate the bone density. Body weight of the test animals and feed intake by the test animals were measured twice a week during the administration period.
- the whole bone density, the cancellous bone density, the cortical bone density, the boundary length of the cortical bone adventitia, the length of the cortical bone endosteum, and the bone strength were measured at the site of ⁇ 0.6 mm from the growth plate of the bone (see FIG. 3A ).
- the whole bone density was significantly increased depending on the fargesin dose.
- the cancellous bone density was also significantly increased, depending on the fargesin dose.
- the bone strength was evaluated by using Polar coordinates strength (SSI). As shown in FIG. 15 , there was the trend that the bone strength was improved, depending on fargesin dose.
- SSI Polar coordinates strength
- fargesin has the effects on the bone loss of the young age animals caused by RANKL administration thorough dose-dependent increase of the bone density.
- Bleeding examples of the food comprising fargesin or 90% MeOH fraction are shown in below. Each blending example may be used for producing the functional food or the health foods.
- Composition 1 100 Mannitol 123 Starch 33 Crospovidone 12 Microcrystalline cellulose 30 Magnesium Stearate 2
- compositions were respectively weighted, and homogenously mixed. Then, 300 mg of the mixture was compressed to form a tablet.
- composition 1 40 lactose 150 starch 70 Polyvinylpyrrolidone 5 Crystalline cellulose 35
- compositions were respectively weighted, and homogenously mixed. Then, 300 mg of the mixture was filled with a hard capsule.
- the composition 1 is composed of either fargesin or 90% MeOH fraction, and lactose 1:1. Note that the composition employed in the pharmaceutical preparations 3 to 6 are the same as the composition 1.
- composition 1 100 Tocopherol 0.2
- compositions were respectively weighted, and homogenously mixed. Then, 100 mg of the mixture was filled with a soft capsule.
- Composition 1 200 Lactose 450 Corn starch 300 Hydroxypropyl cellulose 50 Crystalline cellulose 35
- compositions were respectively weighted, and homogenously mixed to produce granule agent by using conventional method.
- compositions were weighed, and both sugar and saccharin were dissolved in 60 ml of distilled water for injection. Then, the composition 2 dissolved in glycerin and ethanol and a solution of seasonings were added to have a mixture. Distilled water was added to the mixture to become final amount to 100 mL to prepare a syrup agent.
- the present invention is useful in the field of production and development for pharmaceutical preparations, functional food, health food and the like.
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Engineering & Computer Science (AREA)
- Food Science & Technology (AREA)
- Polymers & Plastics (AREA)
- Veterinary Medicine (AREA)
- General Health & Medical Sciences (AREA)
- Public Health (AREA)
- Medicinal Chemistry (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Botany (AREA)
- Natural Medicines & Medicinal Plants (AREA)
- Organic Chemistry (AREA)
- Physical Education & Sports Medicine (AREA)
- Inorganic Chemistry (AREA)
- Epidemiology (AREA)
- Mycology (AREA)
- Nutrition Science (AREA)
- Microbiology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Rheumatology (AREA)
- Orthopedic Medicine & Surgery (AREA)
- General Chemical & Material Sciences (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Biotechnology (AREA)
- Alternative & Traditional Medicine (AREA)
- Medical Informatics (AREA)
- Coloring Foods And Improving Nutritive Qualities (AREA)
- Medicines Containing Plant Substances (AREA)
- Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)
- Acyclic And Carbocyclic Compounds In Medicinal Compositions (AREA)
- Heterocyclic Carbon Compounds Containing A Hetero Ring Having Oxygen Or Sulfur (AREA)
Abstract
Provided is a composition that, even when used in a very small amount, has the effect of improving bone density and promoting bone growth, and has few side effects, and also provided are a pharmaceutical preparation for promoting bone growth, functional food product, and health food product containing the composition as an active ingredient. The composition can be obtained from any plant part selected from the group consisting of the bud, leaf, bark or wood of a plant belonging to the family Magnoliaceae. The composition comprises at least one selected from the group consisting of fargesin and physiologically acceptable salts, hydrates, and glycosides thereof. The pharmaceutical preparation, functional food product, and health food product comprising this composition as an active ingredient sufficiently improve bone density and promote bone growth, even when used in small amounts, and therefore realize preventive and/or therapeutic effects against bone disease etc. such as osteoporosis.
Description
- The present invention relates to pharmaceutical composition for osteogenesis promotion comprising axial-equatorial aryl orientational furofuran type lignan, pharmaceutical preparation comprising thereof, functional food comprising thereof and health food comprising thereof. Particularly, the present invention relates to pharmaceutical composition for osteogenesis promotion comprising Fargesin and its derivatives, pharmaceutical preparation comprising thereof, functional food comprising thereof and health food comprising thereof.
- Recently, bone disease patient number of aged people is increasing, depending on the increase of a mean age. Here, the term “bone disease” includes non-metabolic bone disease such as bone fracture and the like, and metabolic bone disease such as osteoporosis, bone Paget's disease, osteomalacia and the like. Several bone disease are caused by an inflammatory arthritis such as osteoarthritis, rheumatoid arthritis, and the like. Rheumatoid arthritis may cause periarticular site of osteoporosis.
- The metabolic bone disease, osteoporosis, is roughly classified into primary osteoporosis, which is not caused by other disease, and secondary osteoporosis, which is caused by other disease such as malignancy, rheumatoid, and others. In the osteoporosis, the primary osteoporosis accounts for 95% of entire of osteoporosis. Further, osteoporosis has type I of which rate of crises is 6 times higher in women than men, and type II which generally develops in the patient over 60 years old.
- In Type I osteoporosis, osteoclast cells, which plays a role in the bone metabolism, is activated. As a result, bone resorption is enhanced, and bone density is reduced. This is caused by increase of cytokine levels derived from decreased estrogen secretion from ovary. Therefore, β-estradiol, a kind of estrogen, is uses as a prophylaxis and/or treatment agent for osteoporosis.
- The bone fracture as the non-metabolic bone disease is resulting from the large force loading onto the bone having normal strength at one stroke in a healthy person. In contrast, it is resulting from less force loading, which does not cause the bone fracture in the healthy person; onto the bones weaken by cancer, osteoporosis and the like. It is referred to as pathological fracture.
- The bone fracture is caused by repeated load onto the same place from exercise. It is referred to as “stress fracture”. It is said that the stress fracture is sometimes developed on metatarsal bone. As compared to men athletes, women athletes prone to breaking bones than men. As one of the reasons, there is mentioned that incidence rate of osteoporosis is higher in the women athletes than men athletes.
- An index of the metabolic bone disease, abnormal blood calcium level is employed, because released amount of calcium into the blood is increasing after the development of osteoporosis.
- Among the bone disease described above, in order to treat the metabolic bone disease except the bone fracture, active vitamin D3 as a derivative of vitamin D which plays an important role in calcium metabolism, calcitonin and derivative thereof, hormonal agent such as β estradiol and the like, and a variety of calcium compound and preparations are clinically used. It is known that among them, vitamin D3 approaches the osteoclast cells and osteoblast cells, proto cells or precursor cells thereof to promote their proliferation.
- It is not a treatment agent, it is known that fargesin having the following chemical structure (Fargesin; MW=370.4) dose-dependently inhibits tartrate-resistant acid phosphatase activities in mouse monocyte macrophage type cells under RANKL stimulation in the culture, or marrow monocytoid cells in the culture, and it also inhibits the phosphorylation of p38 and I-κB by RANKL stimulation (non-patent document 1). Fargesin is a lignan, and it is classified as the axial-equatorial aryl orientational furofuran type one based on its structure (patent document 2). Fargesin described on the reference is extracted from M. fargesii belonging to Magnoliaceae or Thurber's Magnolia (Magnolia kobus DC. var. borealis Sarg.).
-
- [Patent document 1] WO 1990/013299 A1
- [Patent document 2] JP 2003-522787
-
- [Non-patent document 1] Proceedings of annual meeting of the Japanese Society of Pharmacognosy, vol. 55, p. 212, Compounds inhibits osteoclast cell proliferation derived from Magnolis, Naomi MASE, Bong-Kuen CHOI, Morikazu HASEGAWA, Toshiaki TERUYA, Takayuki YONEZAWA, Byung-Yoon CHA, Kazuo NAGAI, Je-Tae WOO
- [Non-patent document 2] RESEARCH BULLETIN OF HOKKAIDO UNIVERSITY FORESTS, 53(1): 1-28 Extractives of kitakobushi Magnolia kobus DC. Var. borealis Sarg. I, Yun-Geun KIM, Shuji OZAWA, Yoshihiro SANO, and Takashi SASAYA
- Calcitonin and the derivative thereof, the hormone agent such as β-estradiol and the like are used for treating the bone disease. However, these agents sometimes cannot to be administrated to the patient, depending on absorption in vivo or metabolic problem. Also, there is the problem that it lacks predictability, because their receptor levels are highly individual.
- Therefore, there is a need for new preparation to complement the prescription for treating the bone disease, and for new treatment method by using such agents. Also, there is a need for prophylaxis agent to reverse the progress of osteoporosis.
- Since bone strength is increased by exercise load, the common exercise is effective for preventing osteoporosis. However, if an aged person or a person being usually sedentary performs exercise with no warm up or vigorous exercise, they carry a risk to strain their body. Therefore, it is effective to perform the exercise and to take the functional food or health food. In these foods, even more than effectiveness, higher safety more than the pharmaceutical preparations should be saved. Because of such a situation, no side effects are more important. Therefore, ingredients included in them are preferable to produce the effect, if their amount is small.
- The treatment of the bone fracture is performed by surgical operation or reset in principle, except administration of an analgesic agent. Since osteoporosis causes the bone fracture, the treatment of osteoporosis and prevention are necessary to prevent the bone fracture.
- In order to prevent the stress fracture among the bone fracture, appropriate dietary control, training and organizing to take an adequate rest are necessary. Also, it is effective for the prevention of the stress fracture to take the functional foods or health foods to complement them. For these foods, it is important that they do not cause any side effects like those above. Therefore, the composition for them is preferable to show sufficient prophylactic effects in the small amount.
- Accordingly, there are strong needs for osteogenesis improving pharmaceutical preparations having highly predictable effects, particularly, which has high improving effects for the cancellous bone. From the view point of preventive medicine, there is strong social need for the foods comprising the composition.
- The first aspect of the present invention is a pharmaceutical composition for promoting osteogenesis comprising at least a substance selected from the group consisting of a axial-equatorial aryl orientation furofuran type lignin compound shown in the following chemical formula (I), a pharmacologically acceptable salt thereof, a pharmacologically acceptable hydrate thereof, and a pharmacologically acceptable glycoside thereof.
- (In the formula, R1 and R4 independently show one of functional group selected from the group consisting of a hydrogen atom, alkyl group having a
carbon number 1 to 3, hydroxyl group, alkoxy group having thecarbon number 1 to 3; R2 and R3 independently show one of functional group selected from the group consisting of alkyl group having acarbon number 1 to 3.) - The pharmaceutical composition for promoting osteogenesis preferably comprises at least a substance selected from the group consisting of a compound shown in the following chemical formula (II), a pharmacologically acceptable salt thereof, a pharmacologically acceptable hydrate thereof, and a pharmacologically acceptable glycoside thereof.
- The pharmaceutical composition is preferably applied for osteoporosis, hypercalcemia, hyper-parathyroid hormonemia, bone Paget's disease, arthritis, rheumatoid arthritis, metastasis of mammary cancer, osteomalacia, malignancy, and nutrition disorder, traumatic bone fracture, stress fracture or the like. Particularly, it is preferably applied to osteoporosis.
- The second aspect of the present invention is a pharmaceutical composition for promoting osteogenesis comprising an extract fraction obtained from one organ selected from the group consisting of a flower bud, leaf, cortex and xylem of Magnoliaceae plant, of which fraction containing the compound shown in the above-mentioned formula (II).
- The organ selected from the group consisting of the flower bud, leaf, cortex and xylem of Magnoliaceae plant is preferably obtained from the plant selected from the group consisting of Tamushiba (Magnolia salicifolia Maximowicz), Kobushi (Magnolia kobus De Candolle, Magnolia biondii Pampanini, Magnolia sprengeri Pampanini), Hakumokuren (Magnolia heptapeta Dandy (Magnolia denudata Desrousseaux) (Magnoli-aceae), and Kitakobushi (Magnolia praecocissima var. borealis). When the flower bud is used of Kobushi, fractions obtained from it have higher fargesin content.
- The third aspect of the present invention is the pharmaceutical preparation comprising the above-mentioned pharmaceutical composition as the active ingredient to be administrated at a predetermined dosage. In the pharmaceutical preparation, the predetermined dose is preferably 10 to 350 mg/day in compound equivalent, more preferably 20 to 175 mg/day. The pharmaceutical preparation is preferably applied for osteoporosis, hypercalcemia, hyper-parathyroid hormonemia, bone Paget's disease, arthritis, rheumatoid arthritis, metastasis of mammary cancer, osteomalacia, malignancy, and nutrition disorder, traumatic bone fracture, stress fracture or the like. Particularly, it is preferably applied to osteoporosis.
- The fourth aspect of the present invention is the functional food comprising the composition of the first and/or the second aspect at the predetermined content. The fifth aspect of the present invention is the functional food comprising the composition of the first and/or the second aspect at the predetermined content.
- The food is more preferably a functional food or the health food for promoting the osteogenesis. The predetermined content is preferably 1 to 1,000 mg/kg. The amount intake of the functional food or the health food is preferably 10 to 350 mg/day expressed in terms of the amount of the above-mentioned compound, and more preferably 20 to 175 mg/day. The functional food or the health food may be preferably used to the bone disease such as osteoporosis, more preferably used to osteoporosis.
- The food may be cookies and biscuits, wheat and miscellaneous cereals for being supplemented to rice, noodles such as Japanese wheat noodle, soba noodle, and pasta, dairy product such as cheese, yogurt, jam, mayonnaise, processed soy product such as soybean paste, soy source, tea, coffee and cocoa, nonalcoholic beverage such as soft drinks and fruits juice, alcoholic beverage such as medicated liquor, snacks such as candy (drops), and chocolate, chewing gum, Japanese cracker, sweets made from azuki-bean such as azuki-bean jelly, to produce the functional food.
- The sixth aspect of the present invention is a treatment method for osteogenesis comprising the step for non-parenterally or parenterally administrating any one selected from the group consisting of the composition of the first aspect of the present invention, that of the second aspect of the present invention, and the pharmaceutical preparations to the patient necessary for promoting the cortical bone formation or the cancellous bone formation.
- Wherein, the composition or the pharmaceutical preparation is preferably taken orally; more preferably, it is combined with the exercise therapy, because fixing ration of calcium to the bone is improved.
- Small amounts of the composition, pharmaceutical preparation or foods, or the active ingredients or compositions used in the treatment method have sufficient advantageous effects for the bone density enhancement or the bone growth acceleration, and another advantageous effect to prevent and/or treat the bone diseases. Therefore, they have few side effects.
-
FIG. 1 is a graph showing 1H NMR specturm (400 MHz, CDCL3) of fargesin. -
FIG. 2 is the graph showing 13C NMR spectrum (400 MHz, CDCL3) of fargesin. -
FIG. 3A shows measurement site in a femur. -
FIG. 3B shows a section of the measurement site in the femur. -
FIG. 4 is the graph showing a bone density (mg/cm3) of the total bone, when a test substance is administrated to an ovariectomized mouse. -
FIG. 5 is the graph showing a bone density (mg/cm3) of the cancellous bone, when a test substance is administrated to an ovariectomized mouse. -
FIG. 6 is the graph showing a bone density (mg/cm3) of the cortical bone, when a test substance is administrated to an ovariectomized mouse. -
FIG. 7 is an image of transmitted light of the bone (left column) and a fluorescence staining image of the bone by using calcein (right column). -
FIG. 8A is the graph showing ALP activities and TRAP activities by using a relative ratio to a negative control (%), when the test substance is administrated to an ovariectomized mouse. -
FIG. 8B is a double stained image of ALP and TRAP, when the substance is administrated to co-cultured cells. -
FIG. 9A is the graph showing ALP activities and results of MTT assay by using the relative ratio the negative control (%), when the test substance is administrated to osteoblast-like cells. -
FIG. 9B is the graph showing ALP stained image, when the test substance is administrated to the osteoblast-like cells. -
FIG. 10A is the graph showing ALP activities by using the relative ratio the negative control (%), when the test substance is administrated to calcified osteoblast-like cells. -
FIG. 10B is the graph showing ALP stained image, when the test substance is administrated to the calcified osteoblast-like cells. -
FIG. 10C is the graph showing stained image of mineral deposition, when the test substance is administrated to the osteoblast-like cells. -
FIG. 11A is the graph showing the bone density in the mice of each group, a pseudo-operation group (Sham), or an ovariectomized group (OVX), after 3 month from the administration, when each substance was administrated to the mice. -
FIG. 11B is a figure showing the cancellous bone density of mice in the groups in shown inFIG. 11A . -
FIG. 12 is a figure showing polar coordination strength of mouse femur in the groups shown inFIG. 11A . -
FIG. 13 is a figure showing values TRACP5b of mouse sera of the groups shown inFIG. 11A . -
FIG. 14A is a graph showing the total bone density of the mice of the sRANKL administration or the test sample administration group after 13 days from administration. -
FIG. 14B is the graph showing the mouse cancellous bone density of the group shown inFIG. 14A . -
FIG. 15 is the graph showing the polar coordination strength of mouse femur in the groups shown inFIG. 14A . - The present invention is explained in detail hereinbelow.
- The first aspect of the present invention is a pharmaceutical composition for promoting osteogenesis comprising at least a compound shown in the following formula (I), the axial-equatorial aryl orientational furofuran type lignin compound and their derivatives.
- In the formula, R1 and R4 independently show one of functional group selected from the group consisting of a hydrogen atom, alkyl group having a
carbon number 1 to 3, hydroxyl group, alkoxy group having thecarbon number 1 to 3; R2 and R3 independently show one of functional group selected from the group consisting of alkyl group having acarbon number 1 to 3. - The compound shown in the formula (I) is preferably that sown in the formula (II), because it has high promoting activities of bone density, growth of bones, formation of the cortical bone or cancellous bone.
- Wherein, the derivative includes the physiologically acceptable salt thereof, hydrate thereof, and a glycoside thereof, and a mixture thereof. As the physiologically acceptable salt thereof, there are mentioned such as sodium salt, potassium salt, chloride salt and the like. Also, as the physiologically acceptable hydrate, there are mentioned such as monohydrate, dehydrate and the like.
- The compound shown in the formulae (I) to (III) and analogs thereof, salt thereof, hydrate thereof, glycoside thereof, and mixture thereof may be prepared by using the conventional method or according to that to obtain. Commercially available ones may be purchased.
- For example, the composition of the present composition may be produced as described below.
- Firstly, an organ selected from the group consisting of the flower bud, leaves, bark, and xylem is collected to dry to obtain dried ones.
- Concretely, for example, Boushunka, dried flower bud of Magnolia biondii is prepared. The dried flower bud may be prepared to collect them from such plants by using air drying. The commercially available one sold as Shinni as the crude drug may be purchased to use. Instead of the flower but, the leaves, the bark or the xylem may be used.
- The dried one is weighted at a predetermined weight; then about 1.7 to 7 times volume of the weight of methanol is added to perform the extraction at the predetermined temperature. Solid content is separated by filtration from the extract. Then, methanol is removed and the weight of a residue is weighed. Two to five volumes of the residue weight of the mixture of water/ethyl acetate is added to the residue to perform a partitioned extraction at the predetermined temperature.
- When 1 kg of the dried flower bud is used, about 1.7 to 7.0 L of hydrous or non-hydrous alcohol is added, for example, about 0.085 to 70 L of 100% methanol is added to 0.05 to 10 kg of the dried flower bud, to perform the extraction at 3 to 14 days at 2 to 6 degree centigrade.
- From the obtained extract, the solid content is separated by using a device such as Buchner funnel and the like. Subsequently, solvent is removed by using a rotary evaporator, flush evaporator and the like.
- The amount of the residue is weighed, and then, for example, about 2 to 5 time volume of the mixture of water/organic solvent is added to be transferred into a separatory funnel. After that, it is subjected to the partitioned extraction at room temperature.
- For the partitioned extraction, in addition to the partitioned funnel, a liquid-liquid extraction equipment, counter-current extraction equipment, and the like may be chosen, depending on the volume of the dried flower bud or other dried one. Also, for the partitioned extraction, water/ethyl acetate, water/acetone, water/butanol, and the like may be used. Among them, water/ethyl acetate is preferably used, because it is easier to remove the solvent from the organic phase. Particularly, it is preferable to set the ratio of water/ethyl acetate to 0.5/2 to 2/0.5 from the view point of extraction efficiency, more preferably 1/1.
- After the partitioned extraction, the organic phase is separated from the aqueous phase. Then, the organic solvent of the organic phase obtained is removed by using the evaporator and the like to obtain the first concentrated solution. When the many components are extracted in the organic phase, the aqueous phase is separated from the organic phase. Then, the same organic phase to be separated is added to the organic phase at the same volume, and then separated. The procedure is repeated. By this, the intended compounds are extracted more, and the intended compound is obtained at high efficiency.
- Then, the first concentrated solution is performed to the second partitioned extraction by using the different solvent system. Particularly, the mixture of the organic solvent is added to the first concentrated solution at 2 to 5 times volume of the first concentrated solution to perform the second partitioned extraction at the predetermined temperature. In order to eliminate aliphatic components, the solvent system such as n-hexane/water, n-hexane/methanol and the like is preferably employed. When n-hexane/methanol is employed; aqueous methanol including about 10% of water is preferably employed. If desired, the repeated extraction by using n-hexane has an advantageous effect that it accelerates elimination of the aliphatic components, and makes purification hereinafter easier. After that, obtained MeOH phase is separated similarly that as mentioned above to concentrate to obtain the second concentrated solution.
- Not that the composition for prophylaxis and/or treatment of the bone disease may be produced from the according to the conventional method by removing MeOH to obtain crystalline.
- Next, according to the following procedure, 90% MeOH fraction was purified by using a column chromatography to obtain one of the compounds of interest, fargesin.
- Firstly, for example, an open column of diameter from 5 to 20 cm×length 12.5 or 75 cm is prepared, and 200 to 800 g of silica gel is packed into it. The first solvent is poured into the column to swell the silica gel. After the swelling of the gel, the second concentrated solution is applied on the gel to be fractionated by using a step gradient method to obtain the first fractions. The volume of the fractions may be properly decided, it is preferable to set the fraction as 0.75 to 1.5 L, because of the operability efficiency.
- In the step gradient method employed here, for example, the elution solvent may be sequentially changed in stepwise ethyl acetate: n-hexane=1:9 to 10:0. Finally, for elution of the components adsorbed on the silica gel, 100% MeOH is employed. The content of the intended component may be confirmed by using a thin layer chromatography.
- The mixture ratio of the elution solvent to obtain the intended compound of the composition of the present invention is preferably ethylacetate: n-hexane=1:9 to 7:3, more preferably 2:8 to 5:5, far more preferably 3:7. The mixture ratio of the elution solvent to have the highest yield is changed depending on the volume of dried flower bud, quality, solvent volume used for the subsequent extraction operation, the extraction temperature. Therefore, it is preferable to confirm the yield of each fraction by using the thin layer chromatography.
- Note that the crystalline may be sometimes precipitated in the fraction including high content of the intended compound. In this case, the precipitates are separated by the filtration to be recrystallized according to the conventional method to obtain the crystalline with high purity.
- Other fractions are processed as the same as the above-mentioned concentrated solution to obtain the pharmaceutical preparation for osteogenesis promotion of the present invention. The first fractions of them are concentrated as the same as those described above, they are purified the following procedure by using the preparative chromatography.
- For example, the concentrated solution of the first fractions is applied onto a reverse phase column chromatography by using the octadecyl silica column (C18-ODS), the
inner diameter 2 cm×thelength 20 cm; then fractionated by using the preparative chromatography. As the elution solvent, for example, water/methanol, of which mixture ratio are changed by 20%, may be employed. In this case, as the same as the case in which the open column is used, the purification may be performed by using the step gradient method. - The fractions are collected to the same ratio, and they are concentrated, checking the intended compound content. When the intended compounds are included in the 80% methanol fraction to give the precipitate as the crystalline by the concentration, the crystalline may be obtained by filtration of the concentrated fraction with, for example, No. 2 grade filter paper.
- The obtained crystalline is dissolved in the predetermined solvent, and they are subjected to mass spectrometry (MS), nuclear magnetic resonance spectroscopy (NMR). Then, the obtained spectrum data are compared to the reference data to identify the structure of the obtained compound.
- By using thus obtained compound or the composition (partially purified fractions), the pharmaceutical preparation for osteogenesis promotion, the functional food and the health food may be produced.
- Note that the optimal dose of the compounds or the compositions for the human administration is generally about 50 times of that for the mouse. In one example, when it is administrated to the body weight 20 g of the mouse as the dosage of 20 mg/kg body weight/day, or to the body weight 35 g of the mouse at the dosage form of 100 mg/kg body weight/day are respectively converted to the dosage of 20 mg/day or 175 mg/day in human.
- The second aspect of the present invention is the pharmaceutical preparation for osteogenesis promotion comprising the above-mentioned compound as the active ingredient. As the pharmaceutical preparations, there are mentioned non-parenteral dosage from such as injections, suppositories, aerosols, percutaneous form and so forth, parenteral preparations such as tablets, powders, capsules, pills, trochiscus, solutions and so forth. Wherein, the above-mentioned tablet includes sugar coated tablets, coat tablets, and buccal tablets; the capsule includes both of hard capsules and soft capsules. The granules contain coated granules. The above-mentioned solution contains suspensions, emulsions, syrups, elixirs, and so forth, and the syrup includes also dry syrups.
- Other pharmaceutical preparations include the liquid formulation of the above-mentioned compositions, or the gel formulation preparation which is an impregnated gel the liquid form and the like. Note that the above-mentioned formulations include both of the sustained and non-sustained release formulation.
- These preparations may be formulated according to the known pharmaceutical method by using pharmacologically acceptable carrier, excipient, disintegrator, lubricant, colorant, and so forth, for formulating the preparation, described on Japanese Pharmacopoeia.
- As these carriers or excipients, for example, there are mentioned such as lactose, glucose, sucrose, mannitol, potato starch, corn starch, calcium carbonate, calcium phosphate, calcium sulfate, crystalline cellulose, powdered glycyrrhiza extract, powdered gentian, and so forth.
- As the disintegrator, for example, there are mentioned such as starch, agar, powdered gelatin, sodium carboxymethylcellulose, calcium carboxymethylcellulose, crystalline cellulose, calcium carbonate, sodium bicarbonate, sodium alginate and so forth; as the lubricant, for example, there are mentioned such as magnesium stearate, talc, hydrogenated vegetable oil, macrogol and so forth.
- The colorant, which is acceptable to be added to the pharmaceutical preparation, can be used with no limitation. Except these additives, a corrigent and so forth cam be used depending on the necessity.
- When formulating the tablet or the granule, if necessary, they may be coated by using sucrose, gelatin, hydroxypropylcellulose, purified shellac, gelatin, glycerin, sorbitol, ethylcellulose, hydroxy-propylcellulose, hydroxypropylmethylcellulose, polyvinylpyrrolidone, acetate cellulose phthalate, hydroxypropylmethylcellulose phthalate, methylmethacrylate, methacrylate polymer, and so forth to have single coating or plural coatings.
- Furthermore, the capsule can be prepared by encapsulating the granule or powdered preparation into the capsule made of ethylcellulose, gelatin, and so forth.
- When the injection is prepared by using the above-mentioned compound, the physiologically acceptable salt thereof, or the hydrate thereof, a PH regulator, a buffering agent, a stabilizer, a solubilizing agent, and so forth may be added as needed.
- When the preparation for preventing and/or treating bone disease is administrated to a patient, the dosage is depending on conditions such as thickness of the symptom, age, weight, and health status and so forth. In general, the preparation is administrated for an adult in the parenteral or non-parenteral route, at the dosage of 1 mg/kg to 2,000 mg/kg, preferably 1 mg/kg to 1,000 mg/kg once a day or more. In the present invention, it is preferably administrated at the dosage of 10 to 350 mg/day per an adult, and more preferably 20 to 175 mg/day per the adult. Number of administration and amounts a day may be adjusted depending on the conditions described above optionally.
- When the content of the compounds as the active ingredient shown in the above-mentioned formula (I) is less than the lower limit, it does not show sufficient osteogenesis effects. In contrast, when the amount excesses the upper limit, it does not show the effect corresponding to the added amount. (Alternatively, it may cause potential undesirable side effect to a living body being administrated it.)
- Here, in order to form the powdered composition of the present invention, the extract obtained in the production process may be condensed, and dried by using the method such as lyophilization, spray-drying, vacuum-drying and so forth; and then dried extract is pulverized into fine powder. Corn starch, dextrin, cyclodextrin, oyster shell powder may be added as needed
- Alternatively, the above-mentioned binder is optionally added to the powder obtained as described above and compressed to from the tablet. After formulation of the tablet, it may be coated by using the coating agent such as sucrose, gelatin and so forth to formulate the sugar-coated tablet, or coated by other coating agent to formulate enteric coated tablet.
- Furthermore, the powder obtained as describe above may be granulated by using the conventional method to formulate the granule. The powder or granule as mentioned above is encapsulated into capsules in a proper amount to formulate the capsule.
- The above-mentioned compositions are added to, for example, breads, cookies, biscuits, wheat to be supplemented to rice and cereals, noodles such as Japanese wheat noodles, buckwheat noodles, pasta and others, dairy food such as cheese, yogurt and others, jam, mayonnaise, soy bean product such as soy bean paste, soy source and others, nonalcoholic beverage such as tea, coffee and cocoa, soft drinks such as and fruits juice, alcoholic beverage such as medicated liquor, snacks such as candy (drops), and chocolate, chewing gum, Japanese cracker, azuki-bean jelly and so forth, to produce the functional food or healthy food having preventing or treatment effect of periodontal disease.
- Note that the compositions is added to yogurt, soy source, drinks and the like, solubilizing auxiliaries or the stabilizers may be employed not so as to form crystalline of the composition of the present invention to precipitated.
- The composition of the present invention may be employed solely, or as a combination of 2 or more to formulate the powders, the granules, the tablets or the capsules to produce the healthy food.
- The food taken by the bone disease patients for the predetermined period, in predetermined number and amounts promotes the cancellous bone formation of them to effectively prevent the appearance of the bone disease such as osteoporosis and the like.
- The present invention is explained in detail by using examples below, however, the present invention is not limited to them.
- Female Slc: ddy mice of 4 weeks old (Japan SLC, Inc.) was operated with ovariectomy to use as a type I osteoporosis model animal. Also, in order to delete the affection by invasion, a group which is operated without ovariectomy (Sham operation) is set (Sham group). These were the aged model animals.
- They are maintained under the condition of 12 hour light/dark cycle, at temperature of 23±3 degree centigrade, and humidity of 55±5% moisture in TP-102 (Toyo-Rico Co., Ltd.) cages at 4 mice/cage. Animal bedding were changed twice a week and always to use all fresh one, CRF-1 (Oriental Yeast Co., Ltd.) is fed as feed and deionized water as drinking water were freely given. Their body weights were weighed twice a week by the end of the experiment.
- (2-1) Partial Purification of Compounds Derived from Shini
- Thirty five L of methanol is added to 10 kg of Shini (Magnolia kobus (M. praecocissima)) provided in Shan Xi Province in China to immerse to perform extraction at 4 degree centigrade for 7 days. A solid substance was separated by the filtration to obtain filtrate. Then, the whole amount of filtrate was concentrated by using the evaporator to obtain a crud extract.
- Next, the crude extract was poured into 5 L volume of the separatory funnel, and 2 L of water/ethyl acetate (1/1) was added. The funnel was shaken to perform the partitioned extraction. The obtained ethyl acetate phase was concentrated by using the evaporator to be poured into another 5 L of the separatory funnel. Two L of hydrous methanol (10% of water content ratio)/hexane was added into the funnel to perform the partitioned extract again.
- The obtained aqueous methanol phase was concentrated by using the evaporator, and then it was subjected to column chromatography under the following conditions. Step gradient method was employed for elution, by using the following elution buffer to obtain fractions corresponding to each elution buffer (one fraction=2,000 mL). The obtained fractions corresponding to the 30% ethyl acetate and 50% ethyl acetate were used as partially purified Magnolia kobus.
- Column: an opened column with a diameter of 9 cm×a length of 50 cm, filled with a 400 g of Silica gel (BW-820 MH, Fuji Silica Chemical Co.).
Elution buffers: ethyl acetate/hexane (10/90, 20/80, 30/70, 50/50, and 70/30). Since precipitates (crystalline) were formed in the fractions of ethyl acetate/hexane=30/70 and 50/50, they were filtered. The obtained precipitates were 24.5 g. - The precipitates obtained as mentioned above were analyzed by using LC-NMR according to the known method. HPLC conditions were shown in below. Its spectrum of 1H NMR and 13C NMR was measured to determine its chemical structure. Optical rotation was measured to perform their structural analysis.
- HPLC instrument: LC-8020 (Tosoh Corporation)
- Column: Cholester waters φ4.6×250 nm (Nacalai Tesque)
Dissolution Medium: 50% acetonitrile/50% water
Solute temperature: room temperature
Solute concentration: 1 mg/ml
Injection volume: 2 μL
Flow rate: 1 mL/minute
Detective wavelength: UV 215 nm
NMR conditions were as follows:
NMR instrument: JNM-AL-400: FT NMR (400 MHz, JEOL Ltd.)
Solvent: Deuterated chloroform (CDCL3)
Results of the 1H NMR spectrum and the 13C NMR spectrum are shown inFIG. 1 andFIG. 2 , respectively. Since the NMR spectrum data and the optical rotation data were completely correspond to those of fargesin (non-patent document 2), the obtained compound was identified as fargesin. The purity was 98%. - Fargesin was dissolved in 30 mL of a solution (it is referred to as “TD solution”, hereinbelow) containing 4% dimethyl sulfoxide (it is referred to as “DMSO”, hereinbelow) and 4% TWEEN 80 (both were purchased from Wako Pure Chemical Industries, Ltd.) so as that a dosage amount of fargesin becomes 20 mg/kg body wt/day, or 10 mg/kg body wt/day.
- β-
estradiol 4% dimethyl sulfoxide (refer to DMSO thereafter) was dissolved in 30 mL of an aqueous solution containing 2% DMSO to prepare β-estradiol solution so as that the dose amount of it becomes 100 μg/kg body wt/day. - After the surgical operation, the mice were adapted to the circumstances for 5 days, and then they were divided to the Sham group, the negative control, the positive control group, and the test substance administration group (6 mice per group). The ovariectomized mice were used for the negative control group, the positive control group, and the test substance administration group.
- The aqueous solution including 4% DMSO was daily administrated to the mice with Sham operation group and the negative control group (they were collectively referred to as “NC group”) p.o. for 3 months. β-estradiol solution was daily administrated to the ovariectomized mice at the dose of 100 μg/kg body wt/day (it is referred to as “B100 group” thereafter) i.p.
- Fargesin solution was daily administrated to the ovariectomized mice in the test group at the dosage of 20 mg/kg body wt/day (it is referred to as “F20 group”, hereinbelow) or 100 mg/kg body wt/day (it is referred to as “F100 group”, hereinbelow) for 3 month p.o.
- In order to study effects of fargesin on the bone, the femur, the largest long bone, was used.
- The mice in each group were killed by the cervical dislocation under diethyl ether anesthesia. Their right femurs were excised with muscles, and the muscles were removed from the femur after the excision. Lengths of the excised bones from the mice in the groups were measured, and they were dipped in 70% ethanol to be fixed.
- In
FIG. 3 , the bone was shown schematically. The long bone (tubular bone) was composed of two thick and rounded ends (epiphysis), and thin and long part between them (a shaft). The bone extends to longitudinal direction, depending on the growth of a plate-shape epiphyseal cartilage (a growth plate) between the epiphysis and the shaft. The region of 1 mm proximal side from the A position for a measurement was set to that from 1 mm proximal region (epiphysis) from the distal growth plate as decided as the measurement region. - The bone density was measured by using the peripheral quantitative computed tomography (pQCT, XCT Research SA+, Stratec Medizintechnik GmbH, Merk Ltd) (
FIG. 3B ). The measurement was performed under the conditions: a diameter of 90 mm, a voxel size of 0.12 mm, a CT speed of 10 mm/sec, and a block number of 1. Bone mineral content (mg/mm), bone density (mg/cm3), and cross section of the bone (mm2) of the entire of tomographic images (whole bone area) were calculated. - Here, the term “bone mass” means the sum of the bone mineral density and amount of protein substrate. The value of the bone mineral amount divided by the section of the bone is bone mineral amount per unit volume (the bone density).
- Next, the area of the cancellous bone was extracted (peel mode 20), and then the bone mineral amount (mg/mm), the section of the bone (mm2), and the bone density (mg/cm3) were calculated. Further to the cortical bone, the bone mineral amount (mg/mm), the bone density (mg/cm3), the section of the bone (mm2), a bone thickness (mm), the periosteum perimeter of the cortical bone (mm), and the endosteum perimeter of the cortical bone (mm) were calculated.
- Based on the bone diameter and the bone density measured by the pQCT, SSI was calculated. SSI is composed of Polar SSI (torsion strength in polar coordination), X-axis SSI (strength in X axis, three-point bending strength), and Y-axis SSI (Y axis strength, three-point lateral bending strength), and it is expressed by the following general equation.
-
SSI=Z×CBD/ND - Wherein, Z is the coefficient of the cross section (mm3), CBD is the bone density of the cortical bone (mg/cm3), and ND is the physiological bone density (1200 mg/cm3). The coefficient Z is shown as the following equation.
-
Z=(r outer 4 −r inner 4)/r outer×π/4 - router; outer diameter (mm), rinner; inner diameter (mm)
- The measurement sites were as mentioned above. A direction from the proximal epiphysis to the distal epiphysis is defined as the polar direction, X axis direction is defined as a horizontal direction away from the body axis, and Y axis direction is defined as vertically downward direction (
FIGS. 3A and B). The polar coordinate torsion strength, X axis strength, and Y axis strength were respectively calculated. For the analysis of the obtained measurement data, Makejob (Stratec Medizintechnik GmbH., Germany) was used. - Average weights of the mice in each group were 19.8 g to 21.2 g at 4-week-old, 29.2 to 31.5 g at 8-week-old. At 12 week-old, the mice became almost mature and their weights reached 32.1 to 36.4 g. Average weight of B100 group mice was lower than that of NC group mice during mouse growth. However, the average weights of F20 group mice and F100 group mice were equal to that of NC group mice during the mouse growth.
- The bones were classified into the whole bone, the cancellous bone, and the cortical bone (a compact bone), the change of the bone mineral mass, the cross-section area, and the bone density were studied. Results are shown in Tables 1 and 2, and
FIGS. 4 and 5 . In Tables, the number written in lower side in each column shows relative value, when the data of Sham group is 100. The data was statistically processed by using Dennett's two-sided t test to decide whether there is significant difference or not. Each number in the table was shown in average ± standard error. From Tables 3 and after that were the same as Tables 1 and 2. - In the whole bone, the bone density of NC group was significantly lower than that of Sham group. Also, the bone mineral mass of NC group (2.252±0.239 mg/mm) was decreased compared to that of Sham group (3.270±0.234 mg/mm). In the cross sections of both bones, there was no significant difference between these groups. However, the cross section of the bone of Sham group reduced approximately 10% from 5.100±0.266 mm2 to 4.558±0.169 mm2. Thus, it was show that the ovariectomized mouse can be a model animal of Type I osteoporosis.
- On the other hand, the bone mineral mass and bone density were significantly increased in the B100 group compared to those in the Sham group (bone mineral mass 3.325±0.203 mg/mm) to show preventative effects of osteoporosis (Table 1,
FIGS. 4 and 5 ). - In the F20 group, not only the bone mineral mass (3.638±0.164 mg/mm) and the bone density but also the cross section area of the bone (5.643±0.152 mm2) were significantly increased. In the F100 group, not only the bone mineral mass (3.660±0.326 mg/mm) and the bone density but also the bone cross section (5.550±0.351 mm2) were similarly significantly increased to show that bone growth was promoted.
-
TABLE 1 Bone density (mg/cm3) whole bone Cancellous bone Sham 639.450 ± 20.653 (100) 350.750 ± 24.892 (100) Negative control NC 488.460 ± 32.809## (76.4) 216.080 ± 26.302 (61.6) group Positive control B100 652.025 ± 28.386* (102.0) 399.050 ± 53.851 (113.8) group Test group F20 644.367 ± 20.387** (100.8) 431.283 ± 30.506* (123.0) F100 653.300 ± 29.083** (102.2) 460.500 ± 36.222** (131.3) #p < 0.05 against Sham, ##p < 0.005 against Sham *p < 0.05 against NC, **p < 0.005 against NC - Next, the effects of β-estradiol and fargesin on the bone at following regions were studied.
- Compared to Sham group (the bone mineral mass 0.6333±0.077 mg/mm, the cross section areas of the bone 1.787±0.091 mm2, the bone density 350.750±24.892 mg/mm3), those of NC group were as follows: the bone mineral mass was 0.354±0.091 mm2, the cross sectional area of the bone was 1.606±0.058 mm2, and the bone density was 216.080±24.892 mg/mm3, and the trends were the same as those in the whole bone B100 group whose bone mineral mass is 0.725±0.110 mg/mm and the bone density was 399.050±53.851 mg/cm3, and they showed increased trend, but they did not show any significant difference between them.
- In contrast, in F20 group, the bone mineral mass was 0.855±0.067 mg/mm, and the bone density was 431.283±30.506 mg/cm3. In the F100 group, the bone mineral mass was 0.920±0.102 mg/mm and the bone density was 460.500±36.222 mg/cm3. Compared to those of the NC group, the bone mineral mass and the cross section area of both groups were significantly increased (Table 2,
FIGS. 4 and 5 ). Therefore, it was demonstrated that fargesin maintained the bone mineral mass of the cancellous bone, which would be decreased by the ovariectomization, and increase the bone density. - In the Sham group, the bone mineral mass was 2.192±0.180 mg/mm, the cross section area was 2.477±0.182 mm2, the bone density was 822.083±12.665 mg/cm3, and the bone thickness was 0.361±0.023 mg/mm. In contrast, the bone mineral mass was 1.150±0.246 mg/mm, the cross section area of the bone was 1.390±0.277 mm2, the bone density was 826.540±11.518 mg/cm3 and the bone thickness was 0.206±0.040 mg/mm in the NC group. In the NC group, since all of the values were significantly decreased. Particularly, the bone mineral mass, the cross section of the bone, and the bone thickness were severely decreased, it was demonstrated that the cortical bone became thinner and brittler.
- In contrast, the bone mineral mass was 2.163±0.230 mg/mm, the cross section area of the bone was 2.4825±0.230 mm2, the bone density was 867.475±13.724 mg/cm3, and the bone thickness was 0.365±0.038 mg/mm in the F100 group. Compared to those of the NC group, since all of the values were significantly increased, it was demonstrated that β-estradiol had highly effective for preventing brittle cortical bones.
- On the other hand, the bone mineral mass was 2.087±0.160 mg/mm, the cross section area of the bone was 2.427±0.185 mm2, the bone density was 859.483±2.479 mg/cm3, the bone thickness was 0.329±0.026 mg/mm in the F20 group. Compared to those of the NC group, the bone mineral mass, the bone density, and the cross section area of the bone were significantly increased. Also, in the F100 group, the bone mineral mass was 2.240±0.320 mg/mm, the cross section area of the bone was 2.640±0.361 mm2, the bone density was 843.55±8.714 mg/cm3, the bone thickness was 0.370±0.051 mg/mm. Compared to those of the NC group, the bone mineral mass, the bone density, and the cross section area of the bone were significantly increased.
- As described above, it was demonstrated that fargesin had effect for preventing the brittle cortical bone at the same level as that of β-estradiol. Further, it was demonstrated that there were a tendency for fargesin to have was highly effects than those of β-estradiol for boundary lengths of the bone adventia and bone endosteum (Table 2).
-
TABLE 2 Cortical bone Bone Cortical bone lining Bone density thickness adventitia membrane Cortical bone (mg/cm3) (mm) diameter (mm) diameter (mm) Sham 882.083 ± 12.665 0.361 ± 0.023 7.9930 ± 0.207 5.727 ± 0.181 (100) (100) (100) (100) Negative NC 826.540 ± 11.518## 0.206 ± 0.040# 7.5608 ± 0.139 6.301 ± 0.119 control group (93.7) (57.1) (94.6) (110.0) Positive B100 867.475 ± 13.724* 0.365 ± 0.038* 7.9895 ± 0.096 5.699 ± 0.229 control group (98.3) (101.1) (100) (99.5) Test group F20 859.483 ± 2.479* 0.329 ± 0.026 8.4160 ± 0.116* 6.347 ± 0.160 (97.4) (91.1) (105.3) (110.8) F100 843.55 ± 8.714 0.370 ± 0.051* 8.3300 ± 0.278* 6.010 ± 0.313 (95.6) (102.5) (104.2) (104.9) #p < 0.05 against Sham, ##p < 0.005 against Sham *p < 0.05 against NC - Polar axis strength (polar coordinates strength) was 1.599±0.143 mm3 in the Sham group, and 1.145±0.129 mm3 in the NC group. This showed that the bone strength index decreased in the NC group. On the other hand, it was 1.376±0.088 mm3 in the B100 group. There was the tendency for the NC group to decrease the strength; however, there was no significant difference between those groups.
- In contrast, it was 1.615±0.090 mm3 in the F20 group, and was 1.666±0.087 mm3 in the F100 group. In those groups, the bone strength indexes were significantly increased as compared to NC group (Table 3).
- Accordingly, it was demonstrated that fargesin largely increases the bone strength index than β-estradiol does. This shows that fargesin effectively prevents the brittle bone caused by the ovariectomy.
-
TABLE 3 Polar axis bone strength (mm3) Negative control group Sham 1.265 ± 0.147 (71.6)# NC 1.601 ± 0.107 (86.1) Positive control group B100 1.946 ± 0.107 (101.0)* Test group F20 2.005 ± 0.154 (104.2)** F100 1.265 ± 0.147 (71.6)# #p < 0.05 against Sham *p < 0.05 against NC, **p < 0.005 for NC - Eagle's minimum essential mediums (MEM, Life Technologies Japan Corporation (Invitrogen)) was used to prepare the organ culture medium by adding 1% of penicillin/streptomycin (Life Technologies Japan Corporation (GIBCO)), 0.25% of fetal bovine serum (Sigma-Aldrich Co. LLC.), 50 μg/ml of ascorbic acid (Wako Pure Chemical Industries, Ltd.), 1 mM of β-glycerophosphoric acid (Sigma-Aldrich Co. LLC.), and 1 μg/ml of calcein (Sigma-Aldrich Co. LLC.). As a test substance, 0.3 μM of fargesin, or as a control, 0.3% of DMSO (final cone., Wako Pure Chemical Industries, Ltd.) was added to the organ culture medium. Fargesin was used the same solution prepared in Example 1 containing 0.3% DMSO as the final concentration.
- On 15 of pregnancy, mouse fetuses were took from pregnant female ICR mice (Japan SLC, Inc.) by Caesarean operation (E15.5) from the 15th days of a pregnant ICR female mouse by Caesarean section. Left and right metatarsals of the fetus were excised to be placed into the organ culture medium. The organ culture was performed under the conditions of 5% CO2 at 37 degree centigrade for 7 days to observe the effect on long axis direction.
- The long axis of the bone grows depending on that of epiphyseal cartilage existing between an epiphysis and a shaft. When the growth of the epiphyseal cartilage stops, calcium salt resulted in deposition of the substances around the cartilage cells. Then, calcification was occurred from the occification center (ossification). The calcified region was fluorescently-stained by using calcein, and then observed under the microscope. The observed image was shown in
FIG. 7 . - It was observed that the metatarsal was extending tot the long axis direction in the mediums as the increased calcified area by using the fluorescence of the calcein. As compared to the negative control without fargesin (0.3% DMSO addition), the metatarsal extended longer vertically in the medium with 0.3 μM of fargesin. As a result, it was shown that 0.3 μM of fargesin was enough for promoting the bone extension in the tissue concentration.
- Four-week-old ddY male mice (Japan SLC, Inc.) were killed by the cervical dislocation, and then, the long bones of left and right lower extremities were excised with muscles. All of the muscles were removed from the bones, and the femurs and shinbones were obtained. The both ends of the obtained femurs and shinbones were scraped a little by a little. The cells in the bone morrow were pushed out into the following medium by using a 2.5 ml of syringe with a needle (22G×1¼; Thermo corporation). After that, contaminants were eliminated through a filter (70 μM Nylon Cell Strainer; Japan Becton, Dickinson and Company) to obtain more than 2×108 cells of BMCs.
- Osteoblast-like cells, UAMS-32 cells, were purchased from the Institution of Physical and Chemical Research (Japan).
- In order to prepare the basic medium, 10.2 g of α-MEM (powder, Life Technologies Japan Corporation (GIBCO)) was dissolved in 1 L of purified water and 2.2 g/l (w/v) of sodium bicarbonate was added. Then, it was filtered through a sterilized filter of which pore diameter was 0.22 μm (Nihon Millipore K. K). Further, the basic mediums were supplemented with 10% (v/v) fetal bovine serum (FBS) (Sigma, heat-inactivated for 30 minutes at 56° C.).
- In this example, 1 μM of PGE2 and 10 nM of Vitamin D3 (both from Wako Pure Chemical Industries, Ltd.) were added to the medium to prepare a co-culture medium. By using the co-culture medium, cell suspensions of BMCs and UAMS-32 obtained as mentioned above were prepared. For the test group, 2 to 80 μM of fargesin was added, and for the negative control group, DMSO was added so as to be 0.3% of the final concentration.
- BMCs were plated at the concentration of 2×106 cells/well, and UAMS-32 cells were plated at the concentration of 1×105 cells/well in each well of the 96 well plates, respectively. Then, the cells were co-cultured in the co-culture medium under the conditions of 5% CO2 and 37 degree centigrade for 5 days. The medium were changed on the 3rd days from the culture start. After cells were fixed as described in below, alkaline phosphatase (ALP) activity was measured as the index of osteoblast differentiation; tartrate-resistant acid phosphatase activity was measured as the index of osteoclast differentiation.
- The cells were cultured under the above-mentioned conditions, 10% formalin aqueous solution was added into each well still standing for 10 minutes, and then ethanol was added still standing for further 1 minute to fix the cells. In order to measure TRAP activity, 10 mM sodium tartrate/50 mM citric acid buffer solution containing 1.36 mg of p-nitrophenyl sodium phosphate (Sigma-Aldrich Co. LLC.) (pH 4.6) was prepared as a substrate solution for TRAP. The substrate solution for TRAP was added to the well at the volume of 100 μl/well, and reacted for 15 to 20 minutes at a room temperature. The reaction solution was transferred into another 96 well plate, to which 100 μl/well of 0.1N NaOH was previously added to stop the reaction, and they were measured at absorbance of 405 nm.
- 50 mM sodium tartrate/0.1 M of sodium acetate buffer (pH 5.0) including 0.1 mg/ml naphthol AS-MX phosphate (sigma N-4875) and 0.6 mg/ml Fast red violet LB salt (both from Sigma-Aldrich Japan Co. LLC.) was prepared as TRAP staining solution. The TRAP staining solution was added to the wells to stain the cells in negative control group till they took on red color under room temperature. After that, the cells were washed with distilled water. The cells stained red and having not less than two nuclei was decided as multinuclear osteoclast, of which number was counted by using the microscope.
- After the cells were cultured under the conditions, 100 μl/well of methanol cooled to −20 degree centigrade was added into the cells and stood for further 1 minute to fix the cells. ALP substrate solution containing 2.47 mg/ml of 4-nitrophenyl phosphoric acid disodium salt hexahydrate (Sigma-Aldrich Japan Co. LLC.), 2 mM of MgCl2 and 0.1M of Tris-HCL (pH8.5) was prepared.
- ALP substance solution was added with the volume of 100 μl/well, and then the reaction was performed at room temperature for 15 to 20 minutes. Next, the reaction solution was transferred into another 96 well plate being added 100 μl/well of 0.1N NaOH to stop the reaction. Then, the plate was measured the absorbance at 405 nm, and set the value as the index of ALP activity.
- As ALP staining solution, 0.1 M Tris-HCl (pH8.5) containing 0.1 mg/ml Naphthol AS-MX phosphate, 0.02% (v/v) N,N-dimethyl formamide, 0.6 mg/ml of Fast blue BB salt (Sigma F-3378, Sigma-Aldrich Co. LLC.), and 2 mM of MgCl2 was prepared.
- After adding the ALP staining solution, the reaction was performed at room temperature, until the cells of the negative control group were colored blue-violet. Then, the cells were washed. The strength of the staining was determined by visual observation to determine the ALP activity.
- All data was statistically analyzed by using SPSS (registered trademark) Statistics 17.0+Amos 17.0 (S.P.S.S. Jan Inc.) and described by using Average ± Standard Error (SEM). As a test, Dunnett's two-sided t test was employed. Significance level was shown as comparisons between the test group and the negative control group as follows: **: p<0.005, *<0.05. The sample number was more than 3 in each group.
- The absorbance data obtained as described above was shown as the ratio of the activities, when the negative control was 100%.
- Against the negative control group, in the group with 2 to 80 μM fargesin, ALP activities were increased depending on fargesin concentration. However, TRAP activities were decreased in contrast. Particularly, in the group with 60 to 80 μM fargesin, the significant increase of the ALP activities and the significant decrease of the TRAP activities were shown (Table 4 and
FIG. 8A ). This shows that promotion of osteoblast differentiation and inhibition of the osteoclast differentiation were occurred. -
TABLE 4 Concentration (μM) ALP activity (%) TRAP activity (%) 0 100.0 100.0 2 112.6 ± 15.7 89.5 ± 5.4 6 119.8 ± 10.1 88.7 ± 7.2 20 145.6 ± 14.4 74.4 ± 8.5 60 164.3 ± 14.0** 42.4 ± 7.1** 80 198.9 ± 14.6** 35.7 ± 5.6** **p < 0.005 for untreated cells - In the negative control group, both cells stained in red (the osteoclast) and in blue (the osteoclast) were observed. In contrast, in the group with 60 to 80 μM fargesin, the cells differentiated to the osteoclast were rarely observed (
FIG. 8B ). - From the observation results, it was demonstrated that fargesin activated the osteoblast; on the other hand, it inhibited the promotion of the differentiation from the osteoclast precursor cells to the osteoclast by the osteoblast. These facts show the excellent functions of fargesin, which effectively inhibit the increase of the bone resorption and reduction of the bone formation, and improve the balance of the bone reconstruction.
- (1) Test cells and culture conditions
- MC3T3-E1 cells ((IAA) the Institution of Physical and Chemical Research), osteoblast-like cell line derived from the mouse fetus cranial bone cells, were inoculated into the 96 well plate at 4,000 cells/well. As a medium, the basal medium prepared in the Example 3 supplemented with fargesin at 2 to 80 μM was used. Pre-culture was performed by using the basal medium only under the conditions of 5% CO2 and 37 degree centigrade for 3 days. After that, the conditioned medium was changed to fresh medium with fargesin; the culture was performed under the conditions of 5% CO2 and 37 degree centigrade for 3 days. On the
day 4 from the culture start, the conditioned medium was changed to the fresh medium; the culture was further performed under the conditions of 5% CO2 and 37 degree centigrade for 3 days. After the termination of the culture, the following MTT test, measurement of ALP activities, and the staining were performed. - Cell viability was measured by using MTT test. MTT reagent was prepared by dissolving 50 mg of 3-(4,5-dimethyl-thiazol-2-yl)-2,5-diphenyl tetrazolium bromide in 10 ml of PBS (−). After the termination of the culture, the medium containing fargesin was partially removed from each well to adjust the solution volume to 100 μL. Ten μl of the MTT reagent was then added to each well to be reacted to give blue-violet color in the CO2 incubator.
- After the termination of the reaction, all medium was removed from each well, 100 μl of DMSO as a lysis solution was added to each well, and then the absorbance was measured at 570 nm to study the viability of live cells.
- Measurement of the ALP activity, statistical processing of the data, and the observation of differentiated cells were performed as the same way as those in Example 3, except the staining of the osteoclast cells were omitted.
- ALP activity and the cell viability were shown in Table 8 as relative values when the negative control was 100.
- Against the negative control, the group with 20 to 80 μM fargesin did not show any significant difference in the cell viability (Table 5 and
FIG. 9A ). On the other hand, the group with 60 to 80 μM Fargesin showed a trend toward declining the cell viability, compared to the negative control group. In contrast, ALP activity was significantly increased (Table 5 andFIG. 9A ). When 60 to 80 μM fargesin was added, many osteoblast stained in blue were observed (FIG. 9B ). -
TABLE 5 Concentration (μM) ALP activity (%) MTT activity (%) 0 100.0 100.0 2 82.8 ± 8.0 106.1 ± 3.2 6 89.8 ± 9.8 105.4 ± 3.3 20 99.8 ± 13.4 100.1 ± 3.4 60 179.8 ± 31.5* 89.1 ± 3.9 80 167.3 ± 32.7* 92.6 ± 3.7 *P < 0.005 against untreated cells. - From the observation results, it was clearly showed that fargesin activated the osteoblast-like cells even if the level did not any effects on their survival to promote the osteoblast cell function. This means that the fargesin did not cause any side effects and it was useful as the bone formation promotion agent.
- MC3T3-E1 cells as the same as those used in Example 4 were used. In addition, 50 μg/ml of L-ascorbic acid and 10 mM β-Glycerophosphate (both from Sigma-Aldrich Japan Co. LLC.) were added to the basal medium employed in the Example 3 was used. Fargesin prepared in the Example 1 was added to the medium the concentration at 2 to 80 μM.
- MC3T3-E1 was inoculated in each well of the 96 well plates at 4,000 cells/well, and then the plate was pre-cultured by using only basal mediums under the condition of 5% CO2 and 37 degree centigrade for 2 days. After that, the medium was exchanged to the medium containing 50 μg/ml of L-ascorbic acid, 10 mM β-Glycerophosphate, and fargesin, and it was incubated under the condition of 5% CO2 and 37 degree centigrade for 5 days. After the termination of the incubation, the measurement of ALP activity and the differentiated cells were observed as the same as those in Example 3.
- Mineral deposition was stained by using 1% alizarin red observed as the bone calcification (Wako Pure Chemical Industries, Ltd.) according to the conventional method, and then it was observed as the bone calcification.
- ALP activity was shown in Table 6 as relative values, when the negative control group was 100.
- In the group supplemented with 60 to 80 μM fargesin, ALP activity was significantly increased, as compared to the negative control group (Table 6 and
FIG. 10A ). When 60 to 80 μM fargesin were added to the medium, many stained osteoclast cells were observed and ALP activity was higher (FIG. 10B ). Further, when 60 to 80 μM fargesin was added to the medium, an increased mineral deposition side stained in red, namely the increased calcification site was also observed (FIG. 10C ). -
TABLE 2 Concentration (μM) ALP activity (%) 0 100.0 2 93.3 ± 2.6 6 92.8 ± 1.6 20 107.0 ± 3.8 60 173.3 ± 13.7** 80 171.5 ± 12.2** **p < 0.005 against untreated cells - From the results, it was obvious that fargesin promoted the osteoblast maturation and osteoblast calcification. This means that fargesin promotes the mechanical structure of the bone formation through the osteoblast activation. Also, the facts observed in the Example 3 to 5 showed that the effect for the living body of fargesin was supported by molecular mechanism in the cells.
- Four-week-old female Slc; ddy mice (Japan SLC, Inc.) were purchased, and they were divided into 8 groups without acclimate keeping (6 mice per group). They were anesthetized by using 50 mg/kg of pentobarbital to be ovariectomized (it is referred to as “OVX” herein below) or to have Sham operation (it is referred to as “Sham” herein below) under the anesthesia which is given by intraperitoneal administration of 50 mg/kg of Somnopentyl (pentobarbital sodium).
- The reason why they are not kept was to prevent the increase of the weight of white adipose tissue, which causes mistake in treatment.
- From the second day of the ovariectomy (or Sham operation), each group was maintained under the condition of 12 hour light/dark cycle, 23±3 degree centigrade as the room temperature, and humidity 55±5% for 2 months as the bone resorption induction period. The animal bedding was exchanged twice a week and all fresh bedding was used at all times. CRF-1 (Oriental Yeast Co., Ltd.) as the feed and deionized water as drinking water were freely given.
- After two month period from the test start, the bone resorption induction period, the test substance were given to each group at the amount shown in the following Table 7 for 3 month.
- Ninety % of MeOH fraction (Shin-i) obtained in Example 1 and Fargesin (may refer to “Far” thereafter) were respectively dissolved into TD solution at the concentration shown in Table 7, and then they were administrated per os.
- Human PTH (1-34) (herein below, it is sometimes referred to as “hPTH (1-34)”) were dissolved in distilled water at the concentration of 80 μg/kg/day, and it was administrated subcutaneously.
-
TABLE 7 Name of the Administered group solution Route Administration periods Sham OVX TD solvent only p.o. 3 months after the induction period passed Control OVX TD solvent only p.o. The same as above hPTH(1-34) 80 μg/kg/day s.c. The same as above Shini 90% MeOH fr. p.o. The same as above, Fargesin 40 mg/kg/day content corresponds to 2 mg/kg/ day administration group Far 2 2 mg/kg/day p.o. The same as above Far 2020 mg/kg/day p.o. The same as above Far 40 40 mg/kg/day p.o. The same as above - After 3 month from the administration start, the mice belonging to all of the groups were weighed. They were anesthetized by using diethylether, and were collected their blood from their hearts, and then they were died. Then, organs such as the uterus, the white adipose tissue (WAT), the brown adipose tissue (BAT), the liver, the spleen, and both left and right lower extremities were removed. The left and right lower extremities were separated into the femurs and the tibias, and they were stored in 70% EtOH under the room temperature.
- The muscles were removed from the obtained femur (right), and then its bone density and the like were measured by using pQCT employed in Example 1 under the following conditions.
- <Measurement Conditions for pQCT>
- Voxel size (mm): 0.07
- Recognition of contour (CONTMODE): 2 (auto search of ROI)
- PEELMODE: 20
- Recognition method of cancellous bone: Surface & Area
- Ration of cancellous bone area to all of the cross section areas: 35%
- CORTMODE used in CORTBD: 1* * Voxels not larger than the standard value for distinguishing the cortical bone from the cancellous bone (threshold: threshold) were omitted. Each tissue weight was also measured.
- TH value used in CORTBD: 690
- All obtained blood from the heart under diethyl ether anesthesia at dissection was stored at 4 degree centigrade for 24 hours. Then, the blood was centrifuged at 3,000 rpm (4,000×g) for 15 minutes to separate the sera.
- According to the protocol of Mouse TRAP™ Assay (Immuno-diagnostic Systems Ltd, the United of Kingdom), TRACP5b in the obtained sera was determined.
- Among the all data of cancellous bone density obtained from pQCT, both of the highest and the lowest data were deleted to obtain the Median. As to the other parameters were treated as the same as this, the graph was made with N=6. Statistical analysis was performed by using the Dennett's two-sided t test.
- The weights of the body, the uterus, BAT, WAT, the liver, and the spleens of each group were weighed. There were no biases depending on the groups. Also, since the weight gains of the uterus, the inhibition of atrophy, in both of PTH administration group and fargesin administration group were not shown, it was considered that these compounds did not have estrogen-like functions. The amount of feed intake (weight) of Sham group was slightly much than that of the OVX group. However, there was not much difference between them.
- Whole bone mineral content, whole bone densities, and cortical bone mineral content were measured at the site of 1 cm away from the growth plate of the bone. The results were shown in Table 8,
FIGS. 11A and 11B . At the time point of 5 months from the experiment start, the whole bone mineral content and the whole bone densities of the control OVX group were dominantly decreased as compared to those in Sham OVX group. -
TABLE 8 Bone density of the Bone density of Bone density of whole bones cancellous bone cortical bone (mg/cm2) (mg/cm2) (mg/cm2) 5M Sham 525.65 ± 59.21 (100) 116.65 ± 56.36 (100) 983.95 ± 39.93 (100) 5M OVX 381.68 ± 37.65 (72.6)## 35.77 ± 16.96 (30.7)## 931.33 ± 37.78 (94.6) hPTH(1-34) 452.25 ± 49.00 (86.0)* 88.83 ± 30.90 (76.1)** 906.50 ± 32.19 (92.2) 90% MeOH 425.33 ± 38.64 (80.9) 74.90 ± 29.38 (64.2)* 888.52 ± 20.10 (90.3) Far 2 427.83 ± 36.00 (81.4) 64.10 ± 25.48 (54.9)* 911.65 ± 30.15 (92.7) Far 20 437.00 ± 49.48 (83.1) 68.22 ± 21.67 (58.5)* 914.57 ± 43.90 (92.9) Far 40 450.57 ± 33.72 (85.7)** 92.37 ± 41.29 (79.2)* 889.97 ± 23.31 (90.4) ##p < 0.05 against 5M Sham *p < 0.005 against 5M OVX - In the hPTH administration group, both of the whole bone densities and the cancellous bone densities were higher than those in control OVX group. Significant recovery of the bone mass was observed. Particularly, the cancellous bone density was largely increased.
- As shown in
FIG. 11B , the cancellous bone density was dose-dependently increased in the fargesin administration group. As compared to the control OVX group, it was significantly increased in fargesin administration group. The cancellous bone density of 90% MeOH fraction administration group was higher than that ofFar 2 group, and it indicated the possibility of synergic effects with chemical compounds included in the fraction other than fargesin. - The cortical bone mineral content and the cortical bone density were significantly decreased in the Control OVX group compared to that of the Sham OVX group as the same as those of the whole bone and the cancellous bone. The cortical bone content was remarkably decreased. Differently from the cases of the whole bone and the cancellous bone, the bone content was not restored by the administration of PTH. It was also not significantly restored by fargesin administration or 90% MeOH fraction administration (see Table 9).
-
TABLE 9 Perimeter of Bone Perimeter of cortical bone Cross section thickness cortical bone lining area of cortical of cortical adventitia membrane bone (mm2) bone(mm) (mm) (mm) 5M Sham 1.98 ± 0.18 0.27 ± 0.03 8.12 ± 0.19 6.40 ± 0.37 5M OVX 1.36 ± 0.15 0.18 ± 0.03 8.07 ± 0.37 6.93 ± 0.47 PTH 1.69 ± 0.25 0.22 ± 0.03 8.37 ± 0.26 6.98 ± 0.34 90% MeOH 1.40 ± 0.19 0.19 ± 0.03 7.85 ± 0.17 6.64 ± 0.19 Far 2 1.46 ± 0.23 0.20 ± 0.03 8.02 ± 0.26 6.79 ± 0.19 Far 20 1.48 ± 0.18 0.20 ± 0.03 8.04 ± 0.55 6.78 ± 0.68 Far 40 1.56 ± 0.19* 0.21 ± 0.02* 8.14 ± 0.39 6.83 ± 0.38 *p < 0.05 against 5M OVX - As compared to Sham OVX group, the boundary length of cortical bone adventitia was slightly decreased in the Control OVX group. However, that of the cortical bone endosteum was significantly increased. In contrast, both of the boundary length of the cortical bone adventitia and the cortical bone endosteum were significantly increased in PTH administration group. The same trends were shown in fargesin administration group.
- Osteogenesis mainly occurs in the bone adventitia side, and the decrease of the cortical bone mass was appears as extension of Haversian canal (porous formation in the cortical bone). Thus, boundary length of the cortical bone adventitia reflects the osteogenesis (Biomedical Engineering vol. 44, No. 4: 517-521, 2006, ibid. vol. 44, No. 4: 490-502, 2006), and the increase of the length of the cortical bone endosteum reflects the bone resorption. Therefore, it was shown that the bone resorption was increased and the osteogenesis was decreased in the OVX operation group. In contrast, there was the trend that both of the osteogenesis and bone resorption were increased together in PTH administration. It was considered that the bone mass was increased because of dominant osteogenesis. In fargesin administration group, there was the trend that both of the osteogenesis and bone resorption were dose-dependently increased.
- On the other hand, as shown in Table 9, the cancellous bone density was largely increased in 90% MeOH fraction administration group, however, both of the osteogenesis and the bone resorption were not increased.
- Bone strength in the Control OVX group was significantly decreased as compared to that in the Sham OVX group. The bone strengths were significantly increased in both of the PTH administration group and the fargesin administration group (2 mg and 40 mg administration groups) as compared to that of the Control OVX group. The bone strength in the 90% MeOH fraction administration group was higher than that in the Control OVX group; however, there was no significant difference between them (see
FIG. 12 ). - It was known that tartrate-resistant acid phosphatase (TRACP), as a bone metabolism marker, was classified into TRACP 5a and TRACP 5b; TRACP 5a was induced from inflammatory macrophages and TRACP 5b was induced from osteoclast cells, respectively. Circadian change of the TRACP 5b level was low, and it was not affected by nutritional support. Therefore, it was known that the secreted TRACP 5b level indicates the number of osteoclast cells rather than its activity (Name of literatures: Alatalo S L, et al, Clin Chem, 46:1751-1754 (2000)., Alatalo S L, et al, J Bone Miner Res, 18:134-139 (2003)., Chu P, et al, Am J Kidney Dis, 41:1052-1059 (2003)., Alatalo S L, et al, Clin Chem, 50:883-890 (2004).).
- Since TRACP 5b in the sera of the Control OVX group showed remarkably higher level compared to that of the Sham OVX group, it was considered that osteoclast cells had higher activity and large cell number in the Control OVX group (see
FIG. 13 ) - Large fluctuation of TRACP 5b value is not seen in hPTH administration group as compared to OVX group, indicating interrelated perimeters of the cortical bone lining membranes. TRACP 5b value in 90% MeOH fraction administration is higher than that in
Far 2 group, indicating interrelated perimeters of the cortical bone lining membranes. Serum TRACP5b in Fargesin administration group is decreased, showing concentration dependent and is significantly decreased in 40 mg administration group. Thus, it shows that Fargesin restrains the osteoclast from activating and increasing the number, which is the same result as RAW264.7 (FIG. 13 ). - Compared to the OVX group, the level of TRACP 5b did not show major alteration in hPTH administration group; however, it showed the correlation with the length of the cortical bone endosteum. TRACP 5b level in the 90% MeOH fraction administration group was higher than that of Far2 group, and it showed the correlation with the length of the cortical bone endosteum. In fargesin administration group, the level of TRACP5b in the sera was dose-dependently decreased, and it was significantly decreased in the 40 mg administration group. As a result, it was demonstrated that fargesin inhibited the activity of the osteoclast cells and the cell number, similarly to the case in which RAW264.7 was employed (see
FIG. 13 ). - As mentioned above, it was confirmed that fargesin increased the bone densities of the whole bone and the cancellous bone. The measurement results of the boundary length of the cortical bone adventitia, the cortical bone endosteum, and serum TRACP5b activity indicated that fargesin inhibited the osteoclast cell activity and cell numbers, thereby decreasing the capability of the bone resorption to have beneficial osteogenesis effects.
- (1) Test animal
- C57BL/6NCrlCrlj mice (6-week-age, female) were purchased from Oriental Yeast Co., Ltd. and kept to be acclimatized under the conditions of 12 hour light/dark cycle, 23±3 degree centigrade, and humidity 55±5% for 7 days. Two mice were in a cage, and the animal bedding was changed twice a week, and all fresh animal bedding was used at all times. As the feed, CRF-1 (Oriental Yeast Co., Ltd.), and deionized water as the drinking water were freely taken.
- After the acclimatization, these mice were randomly divided into 4 groups (8 mice per group), sRANKL administration group and fargesin administration groups. Fargesin administration groups had 0.2 mg/kg/day administration group (it is referred to as “Far 0.2”, hereinbelow), 2 mg/kg/day administration group (it is referred to as “Far 2” herein below), and 20 mg/kg/day administration group (it is referred to as “Far 20” herein below). sRANKL (1 mg/kg/day, i.p) was administered to all mice on the first day and the second day from test start to cause osteopenia experimentally.
- From the 4th day to 13th day after the test start, distilled water or the test compounds shown in the following Table 10 were daily administered to the mice in each group.
-
TABLE 10 Name of Test solution Groups administrated groups administrated Route Control group RANKL RANKL water solution p.o. (1 mg/kg/day) hPTH administrated Human PTH hPTH(1-34) water solution s.c. group (1-34) (80 μg/kg/day) p.o. Test substance Fargesin 0.2 TD solution of fargesin p.o. administrated group (0.2 mg/kg/ day) Fargesin 2TD solution of fargesin p.o. (2 mg/kg/day) Fargesin 20TD solution of fargesin p.o. (20 mg/kg/day) - After the administration period was finished and the experiment was completed, the femurs were removed in the same way as employed in Example 6 to be excised to evaluate the bone density. Body weight of the test animals and feed intake by the test animals were measured twice a week during the administration period.
- The experiments were performed as the same as done in Example 6, right femur chosen from the obtained femurs was used for measurement of the bone density.
- Results from the measurements of the body weights of each group did not show deviation depending on the groups.
- The whole bone density, the cancellous bone density, the cortical bone density, the boundary length of the cortical bone adventitia, the length of the cortical bone endosteum, and the bone strength were measured at the site of −0.6 mm from the growth plate of the bone (see
FIG. 3A ). - As shown in
FIGS. 14A and 14B , the whole bone density was significantly increased depending on the fargesin dose. The cancellous bone density was also significantly increased, depending on the fargesin dose. - The bone strength was evaluated by using Polar coordinates strength (SSI). As shown in
FIG. 15 , there was the trend that the bone strength was improved, depending on fargesin dose. - As mentioned above, it was demonstrated that fargesin has the effects on the bone loss of the young age animals caused by RANKL administration thorough dose-dependent increase of the bone density.
-
TABLE 11 Bone density Bone density of the of the Bone density of whole cancellous bone cortical bone bone (mg/cm3) (mg/cm3) (mg/cm3) RANKL 486.60 ± 14.10 (100.0) 259.91 ± 23.32 807.66 ± 5.59 (100.0) (100.0) Far 0.2 484.33 ± 11.09 (99.5) 261.64 ± 17.92 797.40 ± 6.12 (100.7) (98.7) Far 2 487.30 ± 16.36 (99.5) 270.30 ± 20.64 798.60 ± 4.56 (104.0) (98.9) Far 20 532.69 ± 8.06 (110)* 336.25 ± 13.50 796.33 ± 3.89 (129.4)* (98.6) *p < 0.05 against RANKL - Bleeding examples of the food comprising fargesin or 90% MeOH fraction are shown in below. Each blending example may be used for producing the functional food or the health foods.
-
-
TABLE 12 Compositions wt % Sugar 53.0 Gum base 20.0 Glucose 10.0 Starch syrup 16.0 Spice 0.5 Composition of the 0.5 present invention Total 100.0 -
-
TABLE 13 Composition wt % Reduced sugar syrup 40.0 Granulated sugar 20.0 glucose 20.0 gelatin 4.6 Water 9.7 Orange fruit juice 4.0 Orange flavor 0.7 Composition of the 1.0 present invention Total 100.0 -
-
TABLE 14 Composition wt % Sugar 50.0 Starch syrup 33.0 Water 14.4 Organic acid 2.0 flavoring 0.2 Composition of the 0.4 present invention Total 100.0 -
-
TABLE 15 Composition Wt % Milk 41.5 Powdered skim milk 5.8 Sugar 8.0 Agar 0.15 gelatin 0.1 lactobacillus 0.005 Composition of the 0.4 present invention flavoring trace Water residue Total 100.0 -
-
TABLE 16 Composition wt % Brown rice germ oil 87.0 Emulsifier 12.0 Composition of the 1.0 present invention Total 100.0 -
-
TABLE 17 Composition Wt. % Roasted coffee beans 6.0 Sugar 6.0 Sodium bicarbonate 0.2 Emulsifier 0.15 Composition of the 1.0 present invention Water residue Total 100.0 -
-
TABLE 18 Composition Wt. % Instant coffee 90.0 Skim milk 7.0 Composition of the 3.0 present invention Total 100.0 -
-
Composition Wt. % Fructose glucose syrup 30.0 Emulsifier 0.5 Flavor Appropriate amount Composition of the 1.0 present invention Purified water residue Total 100.0 -
-
TABLE 20 Composition Wt. % Sugar 76.4 Glucose 19.0 Sucrose fatty acid ester 0.2 Composition of the invention 0.5 Purified Water residue Total 100.0 - Next, the pharmaceutical preparation comprising the composition was shown in below. However, the present invention is not limited to the examples.
-
-
TABLE 21 Component Usage (g) Composition 1100 Mannitol 123 Starch 33 Crospovidone 12 Microcrystalline cellulose 30 Magnesium Stearate 2 - The compositions were respectively weighted, and homogenously mixed. Then, 300 mg of the mixture was compressed to form a tablet.
-
-
TABLE 22 Component Usage (g) Composition 140 lactose 150 starch 70 Polyvinylpyrrolidone 5 Crystalline cellulose 35 - The compositions were respectively weighted, and homogenously mixed. Then, 300 mg of the mixture was filled with a hard capsule. Here, the
composition 1 is composed of either fargesin or 90% MeOH fraction, and lactose 1:1. Note that the composition employed in thepharmaceutical preparations 3 to 6 are the same as thecomposition 1. -
-
TABLE 23 Component Usage (g) Composition 1100 Tocopherol 0.2 - The compositions were respectively weighted, and homogenously mixed. Then, 100 mg of the mixture was filled with a soft capsule.
-
-
TABLE 24 Component Usage (g) Composition 1200 Lactose 450 Corn starch 300 Hydroxypropyl cellulose 50 Crystalline cellulose 35 - The compositions were respectively weighted, and homogenously mixed to produce granule agent by using conventional method.
-
-
TABLE 25 Component Usage (g) Composition 12 Saccharin 0.6 Sugar 30 glycerin 5 Seasoning 0.1 96% ethanol 10.42 Purified water Amount to final volume 100 ml - The above-mentioned compositions were weighed, and both sugar and saccharin were dissolved in 60 ml of distilled water for injection. Then, the
composition 2 dissolved in glycerin and ethanol and a solution of seasonings were added to have a mixture. Distilled water was added to the mixture to become final amount to 100 mL to prepare a syrup agent. -
-
TABLE 26 Component Usage (g) Composition 1100 Calcium silicate 100 - The present invention is useful in the field of production and development for pharmaceutical preparations, functional food, health food and the like.
Claims (12)
1. A pharmaceutical composition for promoting osteoblast differentiation comprising at least a substance selected from the group consisting of a compound shown in the following chemical formula (I), a pharmacologically acceptable salt thereof, a pharmacologically acceptable hydrate thereof, and a pharmacologically acceptable glycoside thereof.
(In the formula, R1 and R4 independently show one of functional group selected from the group consisting of a hydrogen atom, alkyl group having a carbon number 1 to 3, hydroxyl group, alkoxy group having the carbon number 1 to 3; R2 and R3 independently show one of functional group selected from the group consisting of alkyl group having a carbon number 1 to 3.)
2. The pharmaceutical composition for promoting osteoblast differentiation according to the claim 1 , comprising at least a substance selected from the group consisting of a compound shown in the following chemical formula (II), a pharmacologically acceptable salt thereof, a pharmacologically acceptable hydrate thereof, and a pharmacologically acceptable glycoside thereof.
3. A pharmaceutical composition for promoting osteoblast differentiation comprising an extract fraction obtained from one organ selected from the group consisting of a flower bud, leaf, cortex and xylem of Magnoliaceae plant, of which fraction containing the compound shown in the above-mentioned formula (II).
4. The pharmaceutical composition for promoting osteoblast differentiation according to the claim 3 , wherein the organ selected from the group consisting of the flower bud, leaf, cortex and xylem of Magnoliaceae plant is obtained from the plant selected from the group consisting of Tamushiba (Magnolia salicifolia Maximowicz), Kobushi (Magnolia kobus De Candolle, Magnolia biondii Pampanini, Magnolia sprengeri Pampanini), Hakumokuren (Magnolia heptapeta Dandy (Magnolia denudata Desrousseaux) (Magnoli-aceae), and Kitakobushi (Magnolia praecocissima var. borealis).
5. A pharmaceutical preparation comprising the pharmaceutical composition for promoting osteoblast differentiation according to the any one of the claims 1 or 3 for administrating a predetermined dose as an active ingredient.
6. The pharmaceutical preparation for promoting osteoblast differentiation according to the claim 5 , wherein the predetermined dose is 10 to 350 mg/day in compound equivalent.
7. A functional food comprising the composition according to any of the claims 1 or 3 .
8. The functional food according to the claim 7 , wherein a content of the composition is 1 to 1,000 mg/100 g.
9. The functional food according to the claim 7 , wherein the functional food is selected from the group consisting of a biscuit, wheat and coarse cereal to be supplemented to rice, noodle such as buckwheat noodle and pasta, dairy product such as cheese and yoghurt, jam, mayonnaise, processed soy product such as soy source, non-alcoholic beverage such as coffee and cocoa, alcoholic beverage such as medicated liquor, snacks such as candy and chocolate, confectionery such as rice cracker and youkan.
10. A healthy food comprising the composition for promoting osteoblast differentiation according to any one of the claims 1 or 3 .
11. The healthy food according to the claim 10 , wherein a content of the composition is 1 to 1,000 mg/100 g.
12. The healthy food according to the claim 10 , wherein the functional food is selected from the group consisting of a biscuit, wheat and coarse cereal to be supplemented to rice, noodle such as buckwheat noodle and pasta, dairy product such as cheese and yoghurt, jam, mayonnaise, processed soy product such as soy source, non-alcoholic beverage such as coffee and cocoa, alcoholic beverage such as medicated liquor, snacks such as candy and chocolate, confectionery such as rice cracker and youkan.
Applications Claiming Priority (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
JPJP2010-157598 | 2010-07-12 | ||
JP2010157598 | 2010-07-12 | ||
PCT/JP2011/065912 WO2012008467A1 (en) | 2010-07-12 | 2011-07-12 | Pharmaceutical composition for promoting osteogenesis containing axial-equatorial aryl-oriented furofuran-type lignan, and pharmaceutical preparation, functional food product, and health food product comprising composition |
Related Parent Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/JP2011/065912 Continuation WO2012008467A1 (en) | 2010-07-12 | 2011-07-12 | Pharmaceutical composition for promoting osteogenesis containing axial-equatorial aryl-oriented furofuran-type lignan, and pharmaceutical preparation, functional food product, and health food product comprising composition |
Publications (1)
Publication Number | Publication Date |
---|---|
US20130253048A1 true US20130253048A1 (en) | 2013-09-26 |
Family
ID=45469464
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US13/739,340 Abandoned US20130253048A1 (en) | 2010-07-12 | 2013-01-11 | Pharmaceutical Composition for Promoting Osteogenesis Containing Axial-equatorial Aryl-oriented Furofuran-type Lignan, and Pharmaceutical Preparation, Functional Food Product, and Health Food Product Comprising Composition |
Country Status (4)
Country | Link |
---|---|
US (1) | US20130253048A1 (en) |
JP (1) | JP6272646B2 (en) |
KR (1) | KR101491595B1 (en) |
WO (1) | WO2012008467A1 (en) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108367040A (en) * | 2015-10-07 | 2018-08-03 | 株式会社冲绳研究中心 | Including lily magnolia flower extract, its isolate or the pharmaceutical composition from the compound wherein detached as the prevention and treatment for promoting periodontium growth and for periodontitis of active constituent |
TWI668008B (en) * | 2017-09-07 | 2019-08-11 | 財團法人生物技術開發中心 | Magnolia extracts, method for preparing the same and use thereof |
Families Citing this family (3)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN103040805B (en) * | 2012-12-28 | 2014-04-09 | 上海中医药大学 | Medical application of fargesin |
WO2017061781A1 (en) * | 2015-10-07 | 2017-04-13 | 최봉근 | Pharmaceutical composition for promoting periodontal tissue growth and for prevention and treatment of periodontitis, containing magnolia flower extract, fraction thereof or compound isolated therefrom as active ingredient |
CN110051661A (en) * | 2019-05-07 | 2019-07-26 | 中国水产科学研究院长江水产研究所 | Fargesin is inhibiting the application in aquatic livestock Streptococcus agalactiae infections |
Family Cites Families (1)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5240922A (en) | 1989-05-05 | 1993-08-31 | Northern Sydney Area Health Service | Fertility enhancement |
-
2011
- 2011-07-12 WO PCT/JP2011/065912 patent/WO2012008467A1/en active Application Filing
- 2011-07-12 JP JP2012524565A patent/JP6272646B2/en active Active
- 2011-07-12 KR KR1020137003498A patent/KR101491595B1/en active IP Right Grant
-
2013
- 2013-01-11 US US13/739,340 patent/US20130253048A1/en not_active Abandoned
Non-Patent Citations (8)
Title |
---|
Gerstenfeld et al. in Journal of Bone and Mineral Research 24(2), 196 - 208 (2009) * |
Goodman and Gilman's The Pharmacological Basis of Therapeutics (Tenth Edition (2001), McGraw Hill, Chapter I, pages 3-4, 6-8 and 10-29 * |
Im et al. Osteoblast Proliferation and Maturation by Bisphosphonates, Biomaterials 25 (2004) 4105-4115 * |
Remington's Pharmaceutical Sciences, pages 1300 - 1301, Eighteenth Edition (1990), Gennaro, A.R. et al. Editors * |
Sai et al. in Japan Society for Bioscience, Biotechnology and Agrochemistry Meeting vol. 2009, page 16 (2009) * |
Sai et al. in Japan Society for Bioscience, Biotechnology and Agrochemistry Meeting vol. 2009, page 16 (2009) (English Translation) * |
The administration of medicines" in Nursing Times at www.nursingtimes.net/nursing-practice/ specialisms/prescribing/the-administration-of-medicines/288560.article# (retrieved from the internet on July 23, 2014) * |
Zhang et al. in Journal of Clinical Investigation 109:1405 – 1415 (2002) * |
Cited By (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CN108367040A (en) * | 2015-10-07 | 2018-08-03 | 株式会社冲绳研究中心 | Including lily magnolia flower extract, its isolate or the pharmaceutical composition from the compound wherein detached as the prevention and treatment for promoting periodontium growth and for periodontitis of active constituent |
EP3360562A4 (en) * | 2015-10-07 | 2018-09-05 | Choi, Bong Keun | Pharmaceutical composition for promoting periodontal tissue growth and for prevention and treatment of periodontitis, containingmagnolia flower |
JP2018534359A (en) * | 2015-10-07 | 2018-11-22 | チェ, ボン クンCHOI, Bong Keun | Pharmaceutical composition for promoting periodontal tissue growth and preventing and treating periodontitis, comprising as an active ingredient a magnolia flower extract, a fraction thereof, or a compound isolated therefrom |
TWI668008B (en) * | 2017-09-07 | 2019-08-11 | 財團法人生物技術開發中心 | Magnolia extracts, method for preparing the same and use thereof |
Also Published As
Publication number | Publication date |
---|---|
WO2012008467A1 (en) | 2012-01-19 |
JP6272646B2 (en) | 2018-02-07 |
KR101491595B1 (en) | 2015-02-11 |
JPWO2012008467A1 (en) | 2013-09-09 |
KR20130050967A (en) | 2013-05-16 |
Similar Documents
Publication | Publication Date | Title |
---|---|---|
US20100105766A1 (en) | Composition for inhibition or prevention of bone density reduction | |
US20130253048A1 (en) | Pharmaceutical Composition for Promoting Osteogenesis Containing Axial-equatorial Aryl-oriented Furofuran-type Lignan, and Pharmaceutical Preparation, Functional Food Product, and Health Food Product Comprising Composition | |
KR102049440B1 (en) | Composition for preventing and improving woman climacterium symptoms comprising extract of Polygonum cuspidatum Sieb. et Zucc. and Cinnamomum cassia Blume | |
JP5038310B2 (en) | Pharmaceutical composition for prevention and / or treatment of bone disease, functional food or health food containing the composition, and pharmaceutical preparation comprising the composition as an active ingredient | |
KR100910622B1 (en) | Functional food comprising extract of Nelumbo nucifera Gaertn for the prevention and amelioration of osteoporosis | |
KR20150083622A (en) | Anti-obesity composition using acanthopanax sessiliflorus and mulberry | |
JP2009184940A (en) | Therapeutic agent | |
KR101318210B1 (en) | Composition comprising Foeniculum vulgare Miller seed extract for preventing or treating osteoporosis | |
KR20160057525A (en) | composition for the prevention and treatment ofosteoporosis containing Acyranthes bidentata Blume and ginseng extract | |
KR20170015446A (en) | composition for the prevention and treatment ofosteoporosis containing Acyranthes bidentata Blume and ginseng extract | |
KR101305555B1 (en) | Composition for treatment and prevention of bone diseases comprising extract of magnoliae flos's active components | |
KR101972657B1 (en) | Composition comprising extract of Angelica decursiva Franchet et Savatieras or compounds isolated therefrom for preventing or treating osteoporosis | |
WO2020122373A1 (en) | Composition comprising glycyrrhiza uralensis extract as active ingredient for prevention, alleviation, or treatment of late-onset hypogonadism syndrome | |
KR100752411B1 (en) | Extract of nelumbo nucifera gaertn having effect to prevent and treat osteoporosis | |
KR102160627B1 (en) | Pharmaceutical composition for preventing or treating bone disease comprising extracts of branches of Hovenia dulcis Thunb | |
KR101320975B1 (en) | Composition for treatment and prevention of bone diseases comprising extract of magnoliae flos's active components | |
KR102658030B1 (en) | Composition for preventing, treating or improving bone disease or menopausal disease comprising demineralized glasswort extract or fractions thereof, and method for preparing the same | |
KR101320974B1 (en) | Composition for treatment and prevention of bone diseases comprising extract of magnoliae flos | |
JP2013245194A (en) | Oral administration composition | |
KR20130127088A (en) | Composition comprising an extract of alisma canaliculatum for preventing and treating hyperlipidemia or artherosclerosis | |
KR20180032765A (en) | A composition for improving, preventing and treating of fatty liver diseases comprising leek extract | |
WO2013176054A1 (en) | Agent containing ascorbic acid derivative, and use for said agent | |
KR20230164597A (en) | Composition for preventing, ameliorating or treating woman menopausal symptom comprising mixture of fermented soybean and fermented lettuce as effective component | |
KR101120961B1 (en) | Composition for Improving Osteoporosis | |
McAlpine | Tea types and their effects on in vitro mineralization and in vivo bone structure and density |
Legal Events
Date | Code | Title | Description |
---|---|---|---|
AS | Assignment |
Owner name: ERINA CO., INC., JAPAN Free format text: ASSIGNMENT OF ASSIGNORS INTEREST;ASSIGNORS:ASAI, MIDORI;LEE, YOUNG-SIL;MASE, NAOMI;AND OTHERS;SIGNING DATES FROM 20130212 TO 20130318;REEL/FRAME:030759/0291 |
|
STCB | Information on status: application discontinuation |
Free format text: ABANDONED -- FAILURE TO RESPOND TO AN OFFICE ACTION |